secretion

Renal Handling of Urea

July 22, 2017 Laboratory Medicine, Nephrology, Physiology and Pathophysiology, Urology No comments , , , , , , ,

Renal Handling of Urate

Urate, an anion that is the base form of uric acid, provides a fascinating example of the renal handling of organic anions that is particularly important for clinical medicine and is illustrative of renal pathology. An increase in the plasma concentration of urate can cause gout and is thought to be involved in some forms of heart disease and renal disease; therefore, its removal from the blood is important. However, instead of excreting all the urate it can, the kidneys actually reabsorb most of the filtered urate. Urate is freely filterable. Almost all the filtered rate is reabsorbed early in the proximal tubule, primarily via antiporters (URAT1) that exchange urate for another organic anion. Further on the proximal tubule urate undergoes active tubular secretion. Then, in the straight portion, some of the urate is once again reabsorbed. Because the total rate of reabsorption is normally much greater than the rate of secretion, only a small fraction of the filtered load is excreted.

Although urate reabsorption is greater than secretion, the secretory process is controlled to maintain relative constancy of plasma urate. In other words, if plasma urate begins to increase because of increased urate production, the active proximal secretion of urate is stimulated, thereby increasing urate excretion.

Given these mechanisms of renal urate handling, the reader should be able to deduce the 3 ways by which altered renal function can lead to decreased urate excretion and hence increased plasma urate, as in gout: 1) decreased filtration of urate secondary to decreased GFR, 2) excessive reabsorption of urate, and 3) diminished secretion of urate.

Urate, and some other organic solutes, although more membrane permeable in the neutral form, are less soluble in aqueous solution and tend to precipitate. The combination of excess plasma urate and low urinary pH, which converts urate to the neutral uric acid, often leads to the formation of uric acid kidney stones.

Renal Handling of Urea

Urea is a very special substance for the kidney. It is an end product of protein metabolism, waste to be excreted, and also an important component for the regulation of water excretion. Urea differs from all the other organic solutes in several significant ways. 1) There are no membrane transport mechanisms in the proximal tubule; instead, it easily permeates the tight junctions of the proximal tubule where it is reabsorbed paracellularly. 2) Tubular elements beyond the proximal tubule express urea transporters and handle urea in a complex, regulated manner.

Urea is derived from proteins, which form much of the functional and structural substance of body tissues. Proteins are also a source of metabolic fuel. Dietary protein is first digested into its constituent amino acids. These are then used as building blocks for tissue protein, converted to fat or oxidized immediately. During fasting, the body breaks down proteins into amino acids that are used as fuel, in essence consuming itself. The metabolism of amino acids yields a nitrogen moiety (ammonium) and a carbohydrate moiety. The carbohydrate goes on to further metabolic processing, but the ammonium cannot be further oxidized and is a waste product. Ammonium per se is rather toxic to most tissues and the liver immediately converts most ammonium to urea and a smaller, but crucial amount to glutamine. While normal levels of urea are not toxic, the large amounts produced on a daily basis, particularly on a high protein diet, represent a large osmotic load that must be excreted. Whether a person is well fed or fasting, urea production proceeds continuously and constitutes about half of the usual solute content of urine.

The normal level of urea in the blood is quite variable, reflecting variations in both protein intake and renal handling of urea. Over days to weeks, renal urea excretion must match hepatic production; otherwise plasma levels would increase into the pathological range producing a condition called uremia. On a short-term basis (hours to days), urea excretion rate may not exactly match production rate because urea excretion is also regulated for purposes other than keeping a stable plasma level.

The gist of the renal handling of urea is the following: it is freely filtered. About half is reabsorbed passively in the proximal tubule. Then an amount equal to that reabsorbed is secreted back into the loop of Henle. Finally, about half is reabsorbed a second time in the medullary collecting duct. The net result is that about half the filtered load is excreted.

pH Dependence of Passive Reabsorption or Secretion

Many of the organic solutes handled by the kidney are weak acids or bases and exist in both, neutral and ionized forms. The state of ionization affects both the aqueous solubility and membrane permeability of the substance. Neutral solutes are more permeable than ionized solutes. As water is reabsorbed from the tubule, any substance remaining in the tubule becomes progressively more concentrated. And the luminal pH may change substantially during flow through the tubules. Therefore, both the progressive concentration of organic solutes and change in pH strongly influence the degree to which they are reabsorbed by passive diffusion through regions of tubule beyond the proximal tubule.

At low pH weak acids are predominantly neutral, while at high pH they dissociate into an anion and a proton. Imagine the case in which the tubular fluid becomes acidified relative to the plasma, which it does on a typical Western diet. For a weak acid in the tubular fluid, acidification converts much of the acid to the neutral form and therefore, increases its permeability. This favors diffusion out of the lumen (reabsorption). Highly acidic urine tends to increase passive reabsorption of weak acids (and promote less excretion). For many weak bases, the pH dependence is just opposite. At low pH they are protonated cations. As the urine becomes acidified, more is converted to the impermeable charged form and is trapped in the lumen. Less is reabsorbed passively, and more is excreted.

Evaluation of Renal Function

May 3, 2017 Clinical Skills, Critical Care, Nephrology No comments , , , , , , , ,

Assessment of kidney function using both qualitative and quantitative methods is an important part of the evaluation of patients and an essential characterization of individuals who participate in clinical research investigations. Estimating of creatinine clearance has been considered the clinical standard for assessment of kidney function for nearly 50 years, and continues to be used as the primary method of stratifying kidney function in drug pharmacokinetic studies submitted to the United States Food and Drug Administration (FDA). New equations to estimate glomerular filtration rate (GFR) are now used in many clinical settings to identify patients with CKD, and in large epidemiology studies to evaluate risks of mortality and progression to stage 5 CKD, that is , ESKD. Other tests, such as urinalysis, radiographic procedures, and biopsy, are also valuable tools in the assessment of kidney disease, and these qualitative assessments are useful for determining the pathology and etiology of kidney disease.

Quantitative indices of GFR or Clcr are considered the most useful diagnostic tools for identify the presence and monitoring the progression of CKD. These measures can also be used to quantify changes in function that may occur as a result of disease progression, therapeutic intervention, or a toxic insult. It is important to note that the term kidney function includes the combined processes of glomerular filtration, tubular secretion, and reabsorption, as well as endocrine and metabolic functions. This thread critically evaluates the various methods that can be used for the quantitative assessment of kidney function in individuals with normal kidney function, as well as in those with CKD and acute kidney injury (AKI). Where appropriate, discussion regarding the qualitative assessment of the kidney function is also presented, including the role of imaging procedures and invasive tests such as kidney biopsy.

Excretory Function

The kidney is largely responsible for the maintenance of body homeostasis via its role in regulating urinary excretion of water, electrolytes, endogenous substances such as urea, medications, and environmental toxins. It accomplishes this through the combined processes of glomerular filtration, tubular secretion, and reabsorption.

The “intact nephron hypothesis” described by Bricker, more than 40 years ago, proposes that “kidney function” of patients with renal disease is the net result of a reduced number of appropriately functioning nephrons. As the number of nephrons is reduced from the initial complement of 2 million, those that are unaffected compensate; that is, they hyper function. The cornerstone of this hypothesis is that glomerulotubular balance is maintained, such that those nephrons capable of functioning will continue to perform in an appropriate fashion. Extensive studies have indeed shown that single-nephron GFR increases in the unaffected nephrons; thus, the whole-kidney GFR, which represents the sum of the single-nephron GFRs of the remaining functional nephrons, may remain close to normal until there is extensive injury. Based on this, one would presume that a measure of one component of nephron function could be used as an estimate of all renal functions. This, indeed, has been and remains our clinical approach. We estimate GFR and assume secretion and reabsorption remain proportionally intact.

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Filtration

GFR is dependent on numerous factors, one of which is protein load. Bosch suggested that an appropriate comprehensive evaluation of kidney function should include the measurement of “filtration capacity.” Recently, the concept of renal function reserve (RFR) has been defined as the capacity of the kidney to increase GFR in response to physiological or pathologic conditions. This is similar in context to a cardiac stress test. The patient may have no hypoxic symptoms, for example, angina while resting, but it may become quite evident when the patient begins to exercise. Subjects with normal renal function administered an oral or intravenous (IV) protein load prior to measurement of GFR have been noted to increase their GFR by as much as 50%. As renal function declines, the kidneys usually compensate by increasing the single-nephron GFR. The RFR will be reduced in those individuals whose kidneys are already functioning at higher-than-normal levels because of preexisting kidney injury or subclinical loss of kidney mass. Thus, RFR ma be a complementary, insightful index of renal function for many individual with as yet unidentified CKD.

Quantification of renal function (excretory) is not only an important component of a diagnostic evaluation, but it also serves as an important parameter for monitoring therapy directed at the etiology of the diminished function itself, thereby allowing for objective measurement of the success of treatment. Measurement of renal function also serves as a useful indicator of the ability to the kidneys to eliminate drugs from the body. Furthermore, alterations of drug distribution and metabolism have been associated with the degree of renal function. Although several indices have been used for the quantification of GFR in the research setting, estimation of Clcr and GFR are the primary approaches used in the clinical arena.

Secretion

Secretion is an active process that predominantly takes place in the proximal tubule and facilitates the elimination of compounds from the renal circulation into the tubular lumen. Several highly efficient transport pathways exist for a wide range of endogenous and exogenous substances, resulting in renal clearances of these actively secreted entities that often greatly exceed GFR and in some cases approximate renal blood flow. These transporters are typically found among the solute-linked carrier (SLC) and ATB-binding cassette (ABC) super families. Overall, the net process of tubular secretion for drugs is likely a result of multiple secretory pathways acting simultaneously.

Reabsorption

Reabsorption of water and solutes occurs throughout the nephron, whereas the reabsorption of most medications occurs predominantly along the distal tubule and collecting duct. Urine flow rate and physicochemical characteristics of the molecule influence these processes: highly ionized compounds are not reabsorbed unless pH changes within the urine increase the fraction unionized, so that reabsorption may be facilitated.

Endocrine Function

The kidney synthesizes and secretes many hormones involved in maintaining fluid and electrolyte homeostasis. Secretion of renin by the cells of the juxtaglomerular apparatus and production and metabolism of prostaglandins and kinins are among the kidney’s endocrine functions. In addition in response to decreased oxygen tension in the blood, which is sensed by the kidney, erythropoietin is produced and secreted by peritubular fibroblasts. Because these functions are related to renal mass, decreased endocrine activity is associated with the loss of viable kidney cells.

Metabolic Function

The kidney perform a wide variety of metabolic functions, including the activation of vitamin D, gluconeogenesis, and metabolism of endogenous compounds such as insulin, steroids, and xenobiotics. It is common for patients with diabetes and stages 4 to 5 CKD to have reduced requirements for exogenous insulin, and require supplemental therapy with activated vitamin D3 or other vitamin D analogs to avert the bone loss and pain associated with CKD-associated metabolic bone disease. Cytochrome P450, N-acetyltransferase, glutathione transferase, renal peptidases, and other enzymes responsible for the degradation and activation of selected endogenous and exogenous substances have been identified in the kidney. The CYP enzymes in the kidneys are as active as those in the liver, when corrected for organ mass. In vitro and in vivo studies have shown that CYP-mediated metabolism is impaired in the presence of renal failure or uremia. In clinical studies using CYP3A probes in ESRD patients receiving hemodialysis, hepatic CYP3A activity was reported to be reduced by 28% from values observed in age-matched controls; partial correction was noted following the hemodialysis procedure.

Measurement of Kidney Function

The gold standard quantitative index of kidney function is a mGFR. A variety of methods may be used to measure and estimate kidney function in the acute care and ambulatory settings. Measurement of GFR is important for early recognition and monitoring of patients with CKD and as a guide for drug-dose adjustment.

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It is important to recognize conditions that may alter renal function independent of underlying renal pathology. For example, protein intake, such as oral protein loading or an infusion of amino acid solution, may increase GFR. As a result, inter- and intrasubject variability must be considered when it is used as a longitudinal marker of renal function. Dietary protein intake has been demonstrated to correlate with GFR in healthy subjects. The increased GFR following a protein load is the result of renal vasodilation accompanied by an increased renal plasma flow. The exact mechanism of the renal response to protein is unknown, but may be related to extra renal factors such as glucagon, prostaglandins, and angiotensin II, or intra renal mechanisms, such as alterations in tubular transport and tubuloglomerular feedback. Despite the evidence of a “renal reserve,” standardized evaluation techniques have not been developed. Therefore, assessment of a mGFR must consider the dietary protein status of the patient at the time of the study.

Measurement of Glomerular Filtration Rate

  • Measurement of the GFR is most accurate when performed following the exogenous administration of iohexol, iothalamate, or radioisotopes such as technetium-99m diethylenetriamine pentaacetic acid (99mTc-DTPA).

A mGFR remains the single best index of kidney function. As renal mass declines in the presence of age-related loss of nephrons or disease states such as hypertension or diabetes, there is a progressive decline in GFR. The rate of decline in GFR can be used to predict the time to onset of stage 5 CKD, as well as the risk of complications of CKD. Accurate measurement of GFR in clinical practice is a critical variable for individualization of the dosage regimens of renal excreted medications so that one can maximize their therapeutic efficacy and avoid potential toxicity.

The GFR is expressed as the volume of plasma filtered across the glomerulus per unit of time, based on total renal blood flow and capillary hemodynamics. The normal values for GFR are 127 +- 20 mL/min/1.73 m2 and 118 +- 20 mL/min/1.73 m2 in healthy men and women, respectively. These measured values closely approximate what one would predict if the normal renal blood flow were approximately 1.0 L/min/1.73 m2, plasma volume was 60% of blood volume, and filtration fraction across the glomerulus was 20%. In that situation the normal GFR would be expected to be approximately 120 mL/min/1.73 m2.

Optimal clinical measurement of GFR involves determining the renal clearance of a substance that is freely filtered without additional clearance because of tubular secretion or reduction as the result of reabsorption. Additionally, the substance should not be susceptible to metabolism within renal tissues and should not alter renal function. Given these conditions, the mGFR is equivalent to the renal clearance of the solute marker:

GFR = renal Cl = Ae / AUC 0>t

where renal Cl is renal clearance of the marker, Ae is the amount of marker excreted in the urine from time 0 to t, and AUC 0>t is the area under the plasma-concentration-versus-time curve of the marker.

Under steady-state conditions, for example during a continuous infusion of the marker, the expression simplifies to

GFR = renal Cl = Ae / (Css*t)

where Css is the steady-state plasma concentration of the marker achieved during continuous infusion. The continuous infusion method can also be employed without urine collection, where plasma clearance is calculated as Cl = infusion rate / Css. This method is dependent on the attainment of steady-state plasma concentrations and accurate measurement of infusatn concentrations. Plasma clearance can also be determined following a single-dose IV injection with the collection of multiple blood samples to estimate area under the curve (AUC 0>∞). Here, clearance is calculated as Cl = dose/AUC. These plasma clearance methods commonly yield clearance values 10% to 15% higher than GFR measured by urine collection methods.

Several markers have been used for the measurement of GFR and include both exogenous and endogenous compounds. Those administered as exogenous agents, such as inulin, sinistrin, iothalamate, iohexol, and radioisotopes, require specialized administration techniques and detection methods for the quantification of concentrations in serum and urine, but generally provide an accurate measure of GFR. Methods that employ endogenous compounds, such as creatinine or cyst, require less technical expertise, but produce results with greater variability. The GFR marker of choice depends on the purpose and cost of the compound which ranges from $2,000 per vial for radioactive for 125I-iothalamate to $6 per vial for nonradiolabeled iothalamate or iohexol.

Inulin and Sinistrin Clearance

Inulin is a large fructose polysaccharide, obtained from the Jerusalem artichoke, dahlia, and chicory plants. It is not bound to plasma proteins, is freely filtered at the glomerulus, is not secreted or reabsorbed, and is not metabolized by the kidney. The volume of distribution of inulin approximates extracellular volume, or 20% of ideal body weight. Because it is eliminated by glomerular filtration, its elimination half-life is dependent on renal function and is approximately 1.3 hours in subjects with normal renal function. Measurement of plasma and urine inulin concentrations can be performed using high-performance liquid chromatography. Sinistrin, another polyfructosan, has similar characteristics to inulin; it is filtered at the glomerulus and not secreted or reabsorbed to any significant extent. It is a naturally occurring substance derived from the root of the North African vegetable red squill, Urginea maritime, which has a much higher degree of water solubility than inulin. Assay methods for sinistrin have been described using enzymatic procedures, as well as high-performance liquid chromatography with electrochemical detection. Alternatives have been sought for inulin as a marker for GFR because of the problems of availability, high cost, sample preparation and assay variability.

Iothalamate Clearance

Iothalamate is an iodine-containing radio contrast agent that is available in both radiolabeled (125I) and nonradiolabeled forms. This agent is handled in a manner similar to that of inulin; it is freely filtered at the glomerulus and does not undergo substantial tubular secretion or reabsorption. The nonradiolabeled form is most widely used to measure GFR in ambulatory and research settings, and can safely be administered by IV bolus, continuous infusion, or subcutaneous injection. Plasma and urine iothalamate concentrations can be measured using high-performance liquid chromatography. Plasma clearance methods that do not require urine collections have been shown to be highly correlated with renal clearance, making them particularly well-suited for longitudinal evaluations of renal function. These plasma clearance methods require two-compartment modeling approaches because accuracy is dependent on duration of sampling. For example, Agarwal et al. demonstrated that short sampling intervals can overestimate GFR, particularly in patients with severely reduced GFR. In individuals with GFR more than 30 mL/min/1.73 m2 (greater than 0.29 mL/s/m2), a 2-hour sampling strategy yielded GFR values that were 54% higher compared with 10-hour sampling, whereas the 5-hour sampling was 17% higher. In individuals with GFR less than 30 mL/min/1.73 m2, the 5-hour GFR was 36% higher and 2-hour GFR was 126% higher than the 10-hour measurement. The authors proposed a 5- to 7- hour sampling time period with eight plasma samples to be the most appropriate and feasible approach for most GFR evaluations.

Iohexol

Lohexol, a nonionic, low osmolar, iodinated contrast agent, has also been used for the determination of GFR. It is eliminated almost entirely by glomerular filtration, and plasma and renal clearance values are similar to observations with other marker agents: Strong correlations of 0.90 or greater and significant relationships with iothalamate have been reported. These data support iohexol as a suitable alternative marker for the measurement of GFR. A reported advantage of this agent is that a limited number of plasma samples can be used to quantify iohexol plasma clearance. For patients with a reduced GFR more time must allotted – more than 24 hours if the eGFR is less  than 20 mL/min.

Radiolabeled Markers

The GFR has also been quantified using radiolabeled markers, such as 125I-iothalamate, 99mTc-DPTA, and 51Cr-ethylenediaminetetraacetic acid. These relatively small molecules are minimally bound to plasma proteins and do not undergo tubular secretion or reabsorption to any significant degree. 125I-iothalamate and 99mTc-DPTA are used in the United States, whereas 51Cr-EDTA is used extensively in Europe. The use of radiolabeled markers allows one to determine the individual contribution of each kidney to total renal function. Various protocols exist for the administration of these markers and subsequent measurement of GFR using either plasma or renal clearance calculation methods. The non renal clearance of these agents appears to be low, suggesting that plasma clearance is an acceptable technique except in patients with severe renal insufficiency (GFR less than 30 mL/min). Indeed, highly significant correlations between renal clearance among radiolabeled markers has been demonstrated. Although total radioactive exposure to patients is usually minimal, use of these agents does require compliance with radiation safety committees and appropriate biohazard waste disposal.

Optical Real-Time Glomerular Filtration Rate Markers

A clinically applicable technique to rapidly measure GFR, particularly in critically ill patients with unstable kidney function, is highly desirable. The currently available GFR measurement approaches, as outlined above, are technically demanding, time-consuming, and often cost-prohibitive. Research is underway to develop rapid, accurate, safe, and inexpensive techniques to address this need.

Creatinine

Although the measured (24-hour) CLcr has been used as an approximation of GFR for decades, it has limited clinical utility for a multiplicity of reasons. Short-duration witnessed mCLcr correlates well with mGFR based on iothalamate clearance performed using the single-injection technique. In a multicenter study of 136 patients with type 1 diabetic nephropathy, the correlations of simultaneous mCLcr, and 24-hour CLcr (compared to CLiothalamate) were 0.81 and 0.49, respectively, indicating increased variability with the 24-hour clearance determination. In a selected group of 110 patients, measurement of a 4-hour CLcr during water diuresis provided the best estimate of the GFR as determined by the CLiothalamate. Furthermore, the ratio of CLcr to CLiothalamate did not appear to increase as the GFR decreased. These data suggest that a short collection period with a water diuresis may be the best CLcr method for estimation of GFR.

A limitation of using creatinine as a filtration marker is that it undergoes tubular secretion. Tubular secretion arguments the filtered creatinine by approximately 10% in subjects with normal kidney function. If the nonspecific Jaffe reaction is used, which overestimates the Scr by approximately 10% because of the noncreatinine chromogens, then the measurement of CLcr is a very good measure of GFR in patients with normal kidney function. Tubular secretion, however, increases to as much as 100% in patients with kidney disease, resulting in mCLcr values that markedly overestimate GFR. For example, Bauer et al. reported that the CLcr-to-CLinulin ratio in subjects with mild impairment was 1.20; for those with moderate impairment, it was 1.87; and in those with severe impairment, it was 2.32. Thus, a mCLcr is a poor indicator of GFR in patients with moderate to severe renal insufficiency, that is, stages 3 to 5 CKD.

Because cimetidine blocks the tubular secretion of creatinine the potential role of several oral cimetidine regimens to improve the accuracy and precision of mCLcr as an indicator of GFR has been evaluated. The CLcr-to-CLDPTA ratio declined from 1.33 with placebo to 1.07 when 400 mg of cimetidine was administered four times a day for 2 days prior to and during the clearance determination. Similar results were observed when a single 800-mg dose of cimetidine was given 1 hour prior to the simultaneous determination of CLcr and CLiothalamate; the ratio of CLcr to CLiothalamate was reduced from a mean of 1.53 to 1.12. Thus a single oral dose of 800 mg of cimetidine should provide adequate blockade of creatinine secretion to improve the accuracy of a CLcr measurement as an estimate GFR in patients with stage 3 to 5 CKD.

To minimize the impact of diurnal variations in Scr on CLcr, the test is usually performed over a 24-hour period with the plasma creatinine obtained in the morning, as long as the patient has stable kidney function. Collection of urine remains a limiting factor in the 24-hour CLcr because of incomplete collections, and interconversion between creatinine and creatine that can occur if the urine is not maintained at a pH less than 6.

Estimating of Glomerular Filtration Rate

Because of the invasive nature and technical difficulties of directly measuring GFR in clinical settings, many equations for estimating GFR have been proposed over the past 10 years. A series of related GFR estimating equations have been developed for the primary purpose of identifying and classifying CKD in many patient populations. The initial equation was derived from multiple regression analysis of data obtained from the 1,628 patients enrolled in the Modification of Diet in Renal Disease Study (MDRD) where GFR was measured using the renal clearance of 125I-iothalamate methodology. A four-variable version of the original MDRD equation (MDRD4), based on plasma creatinine, age, sex, and race, was shown to provide a similar estimate of GFR results when compared to a six-variable equation predecessor. However, this equation was shown to be inaccurate at GFR more than 60 mL/min/1.73 m2, for reasons not associated with standardization of Screening assay results. A recent study conducted by the FDA compared the eGFR estimated by the MDRD4 equation to the CLcr estimated by the Cockcroft-Gault equation in 973 subjects enrolled in pharmacokinetic studies conducted for new chemical entities submitted to the FDA from 1998 to 2010. The MDRD4 eGFR results consistently overestimated the CLcr calculated by the CG method. The FDA investigators concluded that “For patients with advanced age, low weight, and modestly elevated serum creatinine concentration values, further work is needed before the MDRD equations can replace the CG equation for dose adjustment in approved product information labeling.”

A single eGFR equation may not be best suited for all populations, and choice of equation has been shown to impact CKD prevalence estimates. This has led to a revitalized interest in the development of new equations to estimate GFR. The newest equations to be proposed for the estimation of GFR have been derived from wider CKD populations than the MDRD study, and include the CKD-EPI and the Berlin Initiative Study (BIS). The CKD-EPI equation was developed from pooled study data involving 5,500 patients, with mean GFR values of 68 +- 40 mL/min/1.73 m2. It has been reported that the CKD-EPI equation is less biased but similarly imprecise compared to MDRD4.

CKD-EPI Equation

The CKD-EPI study equation was compared to the MDRD equation using pooled data from patients enrolled in research or clinical outcomes studies, where GFR was measured by any exogenous tracer. The results of the study indicated that the bias of CKD-EPI equation was 61% to 75% lower than the MDRD equation for patients with eGFR of 60 to 119 mL/min/1.73 m2. Based on these findings, the CKD-EPI equation is most appropriate for estimating GFR in individuals with eGFR values more than 60 mL/min/1.73 m2. Both KDOQI and the Australasian Creatinine Consensus Working Groups now recommend that clinical laboratories switch from the MDRD4 to CKD-EPI for routine automated reporting. If one’s clinical lab does not automatically calculate eGFR using the CKD-EPI, it becomes a bit of a challenge since the equation requires a more complex algorithm than the MDRD equation.

Limitations of the pooled analysis approach used to develop the MDRD and CKD-EPI equations include the use of different GFR markers between studies, different methods of administration of the GFR markers and different clearance calculations. These limitations may partly explain the reduced accuracy observed with the MDRD4 equation at GFR values more than 60 mL/min/1.73 m2. Additionally, a recent inspection of the MDRD GFR study data showed that large intrasubject variability in GFR measures was a likely contributor to the inaccuracy of the gold standard method that was used to create the MDRD equation.

Cystatin C-Based Equations

Addition of serum cysC as a covariate in equations to estimate GFR has been employed as a means to improve creatinine-based estimations of GFR that historically were limited to the following variables: lean body mass, age, sex, race, and Scr.

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  • Alb, serum albumin concentration (g/dL); BUN, blood/serum urea nitrogen concentration (mg/dL);CKD, chronic kidney disease; cysC, cystatin C; eGFR, estimated glomerular filtration rate; Scr, serum or plasma creatinine (mg/dL).
  • k is 0.7 for females and 0.9 for males, alpha is -0.329 for females and -0.411 for males, min indicates the minimum of Screening/k or 1, and max indicates the maximum of Scr/k or 1.

A significant limitation of serum cysC as a renal biomarker is the influence of body mass on serum concentrations. When using a serum cyst-based estimate of GFR, which incorporates the serum cysC, age, race, and sex, a higher prevalence of CKD was reported in obese patients when compared to the MDRD4 equation. In a recent retrospective analysis of over 1,000 elderly individuals (mean age 85 years) enrolled in Cardiovascular Health Study, GFR was estimated using the CKD-EPI and CKD-EPI-cysC equation, specifically equation 9 in Table e42-6. In this population, all-cause mortality rates were significantly different between equations, suggesting that cysC does not accurately predict mortality risk in patients with low Screening, reduced muscle mass, and malnutrition. The combined use of serum cysC and creatinine in modified CKD-EPI equations has recently been reported. The CKD-EPIcreatinine_cystatin C, equation 10 in Table e42-6 is now recommended for use in patients where unreliable serum creatinine values are anticipated, such as extremes in body mass, diet, or creatinine assay interferences.

Liver Disease

Evaluation of renal hemodynamics is particularly complicated in patients with liver disease and cirrhosis, where filtration fraction is associated with the degree of ascites, renal artery vasoconstriction, and vascular resistance. The estimation of CLcr or GFR can be problematic in patients with preexisting liver disease and renal impairment. Lower-than-expected Scr values may result from reduced muscle mass, protein-poor diet, diminished hepatic synthesis of creatine (a precursor of creatinine), and fluid overload can lead to significant overestimation of CLcr.

Evaluations of new eGFR equations for use in patients with liver disease have yield mixed results. In summary, renal function assessment in patients with hepatic disease should be performed by measuring glomerular filtration, and GFR estimation equations that combine creatinine and cysC are preferred.

Hemostasis Mechanism – Platelet Structure and Function

August 24, 2016 Cardiology, Hematology No comments , , , , , , , , , , , , , , , , , , , , , ,

Platelet Granules and Organelles

Platelets possess secretory granules and mechanisms for cargo release to amplify responses to stimuli and influence the surrounding environment. Platelet granule structures include 𝛼- and dense granules, lysosomes, and peroxisomes. 𝛼-Granules and the dense bodies are the main secretory granules that release cargo (e.g., fibrinogen and adenosine diphosphate [ADP]) upon platelet activation.

Platelet granule secretion begins with a dramatic increase in platelet metabolic activity, set off by a wave of calcium release and marked by increased adenosine triphosphate (ATP) production. After platelet stimulation by agonists, a "contractile ring" develops around centralized granules, the granules fuse with the surface membranes, and then they extrude their contents. Granule secretion in platelets is a graded process that depends on the number, concentration, and nature of the original stimulus/stimuli, either strong (e.g., thrombin and collagen) or weak (e.g., ADP and epinephrine).

𝛼-Granules

What the 𝛼-granules have include:

  • 𝛽-thromboglobulin
  • PF4
  • thrombospondin
  • vWF
  • Fibrinogen
  • Other plasma proteins (small amount)
  • Growth factors
  • 𝛼IIb𝛽3 [platelet receptor]

𝛼-Granules, with a cross-sectional diameter of approximately 300 nm and numbering approximately 50 per platelet, are the predominant platelet granules. They are approximately spherical in shape, with an outer membrane enclosing two distinct intragranular zones that vary in electron density. The larger, electron-dense region is often eccentrically placed and consists of a nucleoid material that is rich in platelet-specific proteins such as 𝛽-thromboglobulin. The second zone, of lower electron density, lies in the periphery adjacent to the granule membrane and contains tubular structures with adhesive GPs such as von Willebrand factor (vWF) and multimerin, along with factor V. Platelets take up plasma proteins and store them in their 𝛼-granules.

Three proteins, 𝛽-thromboglobulin, PF4, and thrombospondin, are synthesized in megakaryocytes and highly concentrated in 𝛼-granules. The first two, 𝛽-thromboglobulin and PF4, show homology in amino acid sequence and share the additional features of localization in the dense nucleoid of 𝛼-granules, heparin-binding properties, and membership in the CXC family of chemokines. Together, they constitute approximately 5% of total platelet protein, and they can serve as useful markers for platelet activation in serum or plasma. Thrombospondin may comprise up to 20% of the total platelet protein released in response to thrombin, and likely participates in multiple biologic prcesses.

vWF is also synthesized by megakaryocytes and is present in the tubular structures of the 𝛼-granule peripheral zone, similar to its localization within Weibel-Palade bodies of vascular endothelial cells. Factor V and multimerin, a factor V/Va-binding protein, co-localize with vWF in platelets but not in endothelial cells. Fibrinogen is also found in 𝛼-granules, but is incorporated actively from plasma and not synthesized by megakaryocytes. In fact, small amounts of virtually all plasma proteins, such as albumin, immunoglobulin G (IgG), fibronectin, and 𝛽-amyloid protein precursor, may be taken up into the platelet 𝛼-granules. 𝛼-Granules also contain many growth factors, including platelet-derived growth factor, transforming growth factor-𝛽1 (TGF-𝛽1), and vascular endothelial growth factor. These signaling molecules may contribute to the mitogenic activity of platelets.

Platelet 𝛼-granules serve as an important reservoir for 𝛼(IIb)𝛽3 that contributes significantly to the surface fibrinogen receptors present on activated platelets. The 𝛼-granule membrane protein, P-selectin (granule membrane protein-140) is translocated to the plasma membrane after platelet activation. Finally, a number of additional proteins have been located to the surface of 𝛼-granules alone, including CD9, platelet endothelial cell adhesion molecule-1 (PECAM-1), Rap 1b, GPIb-IX-V, and osteonectin.

The platelets and megakaryocytes of patients with gray platelet syndrome have decreased numbers of 𝛼-granules and reduced levels of some proteins. It is proposed that there is incorrect targeting of 𝛼-granule proteins to the 𝛼-granule in the megakaryocyte in this disease.

Dense Bodies

Dense bodies contain a large reservoir of ADP, a crtitical agnoist for platelet activation that amplifies the effect of other stimuli. In addition to this nonmetabolic pool of ADP, the  dense bodies are rich in ATP, pyrophosphate, calcium, and serotonin (5-hydroxytryptamine), with lesser amounts of guanosine triphosphate (GTP), guanosine diphosphate (GDP), and magnesium. The adenine nucleotides are synthesized and segregated by megakaryocytes, whereas serotonin is incorporated into dense granules from the plasma by circulating platelets. There is more ADP than ATP in dense bodies, and both can lead to adenosine monophosphate (AMP). In turn, AMP can be dephosphorylated to adenosine or cyclized to produce cyclic AMP, an inhibitor of the platelet-stimulatory response. The dense granule membrane contains P-selectin and granulophysin.

Compared ADP/ATP within the metabolic/cytoplasmic pool (at least two different pools: metabolic pool and cytoplasmic pool), ADP/ATP in storage pool (dense bodies) contains approximately two-thirds of the total platelet nucleotides, mainly in the form of ADP and ATP, and is metabolically inactive, does not rapidly incorporate exogenous adenine or phosphate, and equilibrates slowly with the metabolic pool. Nucleotides in this pool (storage pool) are extruded fromt the platelet during the release reaction and cannot be replenished after release.

The ATP (metabolic pool) that is broken down to provide energy for the release reaction is not rephosphorylated; rather, it is irreversibly degraded to hypoxanthine, which diffuses out of the cell.

Lysosomes

Lysosomes are small, acidified vesicles, approximately 200 nm in diameter, that contain acid hydrolases with pH optima of 3.5 to 5.5, including 𝛽-glucuronidase, cathepsins, aryl sulfatase, 𝛽-hexosaminidase, 𝛽-galactosidase, heparitinase, and 𝛽-glycerophosphatase. Additional protein found in lysosomes include cathepsin D and lysosome-associated membrane proteins (LAMP-1/LAMP-2, which are expressed on the plasma membrane after activation). Lysosomal constituents are released more slowly and incompletely (maximally, 60% of the granules) than 𝛼-granules or dense-body components after platelet stimulation, and their release also requires stronger agonists such as thrombin or collagen.

Organelles

Peroxisomes are rare, small granules, demonstrable with alkaline diaminobenzidine as a result of their catalase activity. The structure may participate in the synthesis of platelet-activating factor.

Mitochondria in platelets are similar, with the exception of their smaller size, to those in other cell types. There are approximately seven per human platelet, and they serve as the site for the actions of the respiratory chain and the citric acid cycle. Glycogen is found in small particles or in masses of closely associated particles, playing an essential role in platelet metabolism.

Platelet Kinetics

Approximately one-third of the total platelet mass appears to pool in the spleen. The splenic pool exchanges freely with the platelets in the peripheral circulation. Administration of epinephrine, which evacuates platelets from the spleen, increases the peripheral platelet count 30% to 50%, and platelet counts in asplenic patients are not affected by epinephrine. Some studies suggest that the splenic pool consists of the youngest, largest platelets. Pathophysiologi states can result in 80% to 90% of platelets being sequestered in the spleen, resulting in thrombocytopenia.

Other organs that have pool of platelets (accounting for about 16% of total platelets) include the lungs and the liver and so on.

The life span of platelet has been estimated to be 8 to 12 days in humans. In steady state, when platelet production equals destruction, platelet turnover has been estmated at 1.2 to 1.5 x 1011 cells per day.

PS: Details of various platelet products can be found in thread "Platelet Transfusion for Patients w/ Cancer" at http://www.tomhsiung.com/wordpress/2013/04/platelet-transfusion-for-patients-with-cancer-part-one/


Platelet Adhesion and Activation

Part I – Adhesion

  • Adhesive ligands: vWF, collagen, fibronectin, thrombospondin, laminin (perphaps)
  • Platelet surface receptors: GPIb/V/IX complex, GPVI, 𝛼IIb𝛽3, 𝛼2𝛽1, 𝛼5𝛽1, 𝛼6𝛽1
  • Interaction of GPVI with collagen activates platelet intergrins
  • At low shear conditions, fibrinogen is the primary ligand (interacting with 𝛼IIb𝛽3), but other ligands may also be involved

Platelet adhesion to exposed subendothelium is a complex multistep process that involves a diverse array of adhesive ligands (vWF, collagen, fibronectin, thrombospondin, and perphaps laminin) and surface receptors (GPIb/V/IX, GPVI, integrins 𝛼IIb𝛽3, 𝛼2𝛽1, 𝛼5𝛽1, and 𝛼6𝛽1). The specific ligand/receptor palyers in primary platelet adhesion are largely dependent on the arterial flow conditions present. In high shear conditions, platelet tethering is dependent on the unique shear-dependent interaction between GPIb/V/IX and subendothelial vWF, derived either from plasma or released by endothelial cells and/or platelets. A tether forms between GPIb and vWF that either halts platelet movement or reduces it such that other interactions can proceed. Subsequent interactions are mediated by GPVI binding to glycineproline-hydroxyproline sites on collagen and perhaps to exposed laminin. The interaction of GPVI with collagen strongly activates platelets such that 𝛼IIb𝛽3, can engage in high-affinity interactions with ligands. At low shear conditions, fibrinogen is thought to be the primary ligand supporting platelet plug formation through its interaction with 𝛼IIb𝛽3, although thrombus formation can take place in the absence of vWF and fibrinogen, so other ligands may also be involved.

Following initial platelet adhesion, subsequent platelet-platelet interactions are intially mediated by two receptors, GPIb/V/IX and 𝛼IIb𝛽3, and their respective contributions are dependent on the flow conditions present. In high shear stress conditions, GPIb/V/IX receptor and vWF ligand action are predominant, with fibrinogen playing a stabilizing role.

Platelet Glycoprotein Ib Complex-von Willebrand Factor Interaction and Signaling

Screen Shot 2016-08-18 at 4.01.30 PMThe interaction of the platelet GPIb "complex" (the polypeptides GPIb𝛼, GPIb𝛽, GPIX, and GPV) with its primary ligand. vWF, is the receptor-ligand pairing that initiates platelet adhesion followed by a cascade of events leading to pathologic thrombosis or physiologic hemostasis. A unique aspect of this receptor-ligand interaction is that it requires the presence of high arterial shear rates to take place, thus explaining the predisposition of platelet-rich "white clots" in the arterial circulation over clots found in the venous circulation, with its relatively lower shear forces, in which clot formation takes place independent of the GPIb complex.

The binding site for vWF is present in the N-terminal 282 residues of GPIb. Important to the interactions are a cluster between residues Asp 252 and Arg 293 containing sulfated tyrosine residues and important anionic residues, a disulfide loop between Cys 209 and Cys 248, and an N-terminal flanking sequence of the leucine-rich repeats (LRG). Mutations involving single amino acid residues within these LRGs account for some cases of the congential bleeding disorder Bernard-Soulier syndrome, in which the GPIb complex binds poorly, or not at all, to vWF.

Unlike other receptors, GPIb does not require platelet activation for its interactions with vWF. In vitro, the interactions of vWF and binding with the GPIb complex occur with generally very low affinity in the absence of shear. The presence of the vancomycin-like antibiotic ristocetin or viper venom proteins, such as botrocetin, promotes the interactions. Mobilization may uncoil vWF to promote interactions with GPIb. The addition of shear, in a parallel-plate flow system, results in platelet interaction with subendothelial vWF that occurs in a biphasic fashion. Likewise, the rate of translocation of platelets from blood to the endothelial cell surface, which is dependent, increases linearly up to wall shear rates of 1,500 s-1, whereas the translocation rate remains relatively constant with the wall shear rate between 1,500 and 6,000 s-1. Thus, the presence of shear is important for promoting the interactions between the GPIb complex and vWF. Studies of real-time thrombus formation in the absence of platelet GPIb complex and in blood from individuals with severe (type 3) von Willebrand disease indicate that GPIb and vWF interaction are required for platelet surface interaction at high shear rates (>1,210 s-1), whereas GPIb deficiency results in poor platelet adhesion at lower shear. Shear accelerates thrombus formation likely by promoting this receptor-ligand interaction.

When the GPIb complex interacts with its vWF ligand under conditions of elevated shear stress, signals are initiated that activate integrin 𝛼IIb𝛽3. The pathways involved lead to a) elevation of intracellular calcium; b) activation of a tyrosine kinase signaling pathway that incorporates nonreceptor tyrosine kinases such as Src, Fyn, Lyn, and Syk, phospholipase C(gamma)2, and adaptor protein such as SHC, LAT, and SLP-76; c) inside-out signaling through the 𝛼IIb𝛽3 integrin followed by platelet aggregation; and d) activation of protein kinase C (PKC), protein kinase G (PKG), and phosphoinositide 3-kinase (PI3K), …… and so on.

Once vWF binds to GPIb-V-IX, signaling complexes form in the vicinity of the GPIb𝛼 cytoplasmic tail consisting of cytoskeletal proteins such as 14-3-3ζ  as well as signaling protein like Src and PI 3-kinase. This process leads to Syk activation, protein tyrosine phosphorylation, and recruitment of other cytoplasmic proteins with pleckstrin homology domains that can support interactions with 3-phosphorylated phosphoinositides and ultimately activation of integrin 𝛼IIb𝛽3.

Glycoprotein Ib Complex Interaction with Thrombin and Other Molecules

The GPIb complex serves as an 𝛼-thrombin binding site on platelets, although the physiologic relevance of the interactions is not clear. The density of GPIb complexes (~20,000/platelet) far exceeds the number of thrombin binding sites reported on platelets (~6,000/platelet). Studies have identified interaction of the GPIb complex with ligands other than vWF. These include a study a reversible association of GPIb with P-selectin, which is examined in more detail in the section "Platelets and Endothelium." The interaction of platelet GPIb with the neutrophil adhesion receptor 𝛼(M)𝛽2 (Mac-1) is discussed in the section "Platelets and White Blood Cells." Additionally, GPIb reportedly interacts with high-moecular weight kininogen, factor XII, and factor XI.

Platelet-Collagen Interaction and Signaling

  • Receptors: GPVI, 𝛼2b𝛽1
  • Ligands: collagens

Collagens, one of the most thrombogenic substance in vessels, are very important activators of platelets in the vascular subendothelium and vessel wall, and thus are prime targets for therapeutic intervention in patients experience a pathologic arterial thrombotic event such as MI or stroke. Platelets have two major surface receptors for collagen, the immunoglobulin superfamily member GPVI and the integrin 𝛼2𝛽1. The former is considered to be the primary palyer in platelet adhesion. In additon to these two surface receptors, the GPIb complex can also be considered an indirect collagen receptor because its subendothelial vWF ligand essentially acts as bridging molecule between platelets and collagen by fixing itself to the latter, which, in turn, acts as scaffolding for the multimers. Collagen adhesion also results in indirect activation of the protease-activated receptor 1 via MMP-1. Other molecules, such as CD 36, may also sustain collagen interaction.

Glycoprotein VI receptor

GPVI is the main receptor involved in collagen-mediated platelet activation. Studies of mice lacking platelet GPVI show that they lose collagen-induced platelet activation due to a defect in platelet adhesion. Thus, GPVI appears to serve as teh initial receptor involved in platelet adhesion, and it activates integrin binding. GPVI alone supports adhesion to insoluble collagens, and works with 𝛼2𝛽1 to promote platelet adhesion to soluble collagen microfibrils. GPVI can also be engaged by collagen-related peptides (arranged in triple helical structures with sequences similar to collagen) and teh snake venom convulxin, which elicit signals through GPVI.

Synergism between GPVI pathways and those related to other adhesion receptors such as GPIb-V-IX and soluble agonists released by activated platelets are likely necessary for the full repertoire of platelet-collagen signaling. Exposure of platelets to collagen surfaces likely results in GPVI clustering that in turn triggers the tyrosine phosphorylation of the FcRγ chain. The GPVI/FcRγ-chain complex leads to platelet activation through a pathway that has many aspects in common with signaling by immune receptors, such as the Fc receptor family and the B- and T-cell antigen receptors.

α2β1 receptor

The first platelet collagen receptor identified was the integrin 𝛼2𝛽1 receptor, also known as platelet GPIa/IIa and lymphocyte VLA-2.

When compared to vWF, collagen is a more efficient substrate when it comes to supporting stable platelet adhesion and thrombus formation. The fact that initial platelet tethering to collagen under high shear flow first requires interaction between vWF and platelet GPIb serves to underscore the importance of the two major collagen receptors, GPVI and 𝛼2𝛽1, in promoting platelet adhesion and activation under shear conditions.

In addition to GPVI, the α2β1 receptor also propagates signaling. The use of α2β1 selective ligands has demonstrated calcium-dependent spreading and tyrosine phosphorylation of several proteins when interaction with platelets takes place.

Physiologic Inhibition of Platelet Adhesion

Negative regulation of platelets is essential to set the stimulus threshold for thrombus formation, determine final clot size and stability, and prevent uncontrolled thrombosis. The mechanisms behind the negative regulation of platelet activation are described later, and in this respect, roles of players such as nitric oxide and prostacyclin have been well characterized. Platelet activation can also be inhibited by signaling through the adhesion moleculde PECAM-1 (CD31). Expressed on a number of blood cells and endothelial cells, PECAM has a wide array of regulatory functions in processes such as apoptosis and cell activation. Following homophilic interactions and/or clustering, PECAM-1 is tyrosine phosphorylated in its cytoplasmic tail ITIM domain. Phosphorylation of PECAM-1 recruits and activates the SH2 domain-containing protein-tyrosine phosphatase, SHP-2. Studies suggest that the PECAM-1/SH-2 complex functions to counteract platelet activating, most particularly for collagen by inhibiting GPVI/FcRγ chain signaling.


Part II – Activation

PAR Thrombin Interactions

  • See Figure 16.9

Platelet thrombin receptors/platelet protease-activated receptors/PARs  and signaling

Screen Shot 2016-08-21 at 12.46.55 PMPARs are G-protein-coupled receptors that use a unique mechanism to convert an extracellular protein cleavage event into an intracellular activation signal. In this case, the ligand is already part of the receptor per se, by virtue of the fact that it is represented by the amino acid sequence SFLLRN (residues 42 through 47) and is unmashed as a new amino terminus after thrombin cleaves the peptide bond between Arg 41 and Ser 42 (Figure 16.9). This "tethered ligand" then proceeds to irreversibly dock with the body of its down receptor to effect transmembrane signaling, as shown in Figure 16.9.

Thrombin signaling in platelet is mediated, at least in part, by four members of a family of G-protein-coupled PARs (PAR-1, -2, -3, and -4). Human platelets express PAR-1 and PAR-4, and activation of either is sufficient to trigger platelet aggregation. PAR-1, -3, and -4 can be activated by thrombin, whereas PAR-2 can be activated by trypsin, tryptase, and coagulation factors VIIa and Xa. Presumably, other proteases are capable  of recognizing the active sites of these receptors and can thus also trigger PAR signaling.

Once activated, PAR-1 is rapidly uncoupled from signaling and internalized into the cell. It is then transported to lysosomes and degraded. Platelet presumably have no need for a thrombin receptor recycling mechanism, becuase once activated, they are irreversibly incorporated into blood clots. Conversely, in cell lines with characteristics similar to megakaryocytes, new protein synthesis is needed for recovery of PAR-1 signaling, and in endothelial cells, sensitivity to thrombin is maintained by delivery of naive PAR-1 to the cell surface from a preformed intracellular pool.

Platelet ADP (Purinergic) Receptors and Signaling

  • P2Y1
  • P2Y12
  • P2X

Evidence that ADP plays an important role in both the formation of the platelet plug and the pathogenesis of arterial thrombosis has been accumulating since its initial characterization in 1960 as a factor derived from red blood cells that influences platelet adhesion. ADP is present in high concentratons (molar) in platelet-dense granules and is released when platelet stimulation takes place with other agonists, such as collagen; thus, ADP serves to further amplify the biochemical and physiologic changes associated with platelet activation and aggregation. Inhibitors of this ADP-associated aggregation include commonly used clinical agents, including ticlopidine, clopidogrel, prasugrel, and ticagrelor, proven to be very effective antithrombotic drugs.

Adenine nucleotides interact with P2 receptors that are ubiquitous among different cell types and have been found to regulate a wide range physiologic processes. They are divided into two groups, the G-protein-coupled superfamily named P2Y and the ligand-gated ion channel superfamily termed P2X. Two G-protein-coupled (P2Y) receptors contribute to platelet aggregation. The P2Y1 receptor initiates aggregation through mobilization of calcium stores, and the P2Y12 receptor is coupled to inhibition of adenylate cyclase and is essential for a full aggregation response to ADP with stabilization of the platelet plug.

PS: ADP >>> P2Y12 >>> inhibition of adenylate cyclase >>> decreased cAMP production >>> decreased intensity of aggregation

Inhibition of either P2Y1 or P2Y12 receptors is sufficient to block ADP-mediated platelet aggregation, and coactivation of both receptors is therefore necessary, through the G proteins Gq and Gi, respectively, for ADP to activate and aggregate the platelet.

Although considered a weak agonist in comparison to collagen or thrombin, ADP clearly palys an important role in thrombus stabilization, likely by contributing to the recruittment of additional platelets to growing thrombi. Aggregation is often reversible when platelets are stimulated by ADP alone. In addition, low concentrations of ADP serve to amplify the effects of both strong and weaker agonists, the latter inlcuding serotonin and adrenaline, among others.

Platelet Activation by Soluble Agonists

Epinephrine

Epinephrine is unique among platelet agonists because it is considered to be  capable of stimulating secretion and aggregation, but not cytoskeletal reorganization responsible for shape change. Platelet responses to epinephrine are mediated through 𝛼2-adrenergic receptors, and these responses have been found to vary among individuals, with some donors with otherwise normal platelets manifesting delayed or absent responses.

Arachidonic acid, thromboxane A2, and thromboxane receptors

After platelet stimulation by a number of agonists, arachidonic acid is generated directly by phospholipase A from its membrane phospholipid precursors (PC, PS, and PI) and indirectly by PLC generation of DAG followed by DAG lipase action. Most platelet agonists are believed to activate this pathway. Three known eicosanoid subsetsl of biochemical compounds are known to be derived from the formation of arachidonic acid – the prostanoids, leukotrienes, and epoxides. Although all three of these pathways are present in platelets, most arachidonic acid ends up being metabolized to thromboxane A2 (TxA2).

TxA2 is produced in platelets from arachidonic acid through the generation of PGH2 by the enzyme cyclo-oxygenase, which is irreversibly inhibited by aspirin through acetylation of a serine residue near its C terminus. PGE2 and PGI2 act to inhibit platelet activation by generating intracellular cAMP, whereas TxA2 activates platelets. Platelets primarily synthesize thromboxane, and endothelial cells mainly synthesize prostaglandins such as PGI2.

Like ADP and epinephrine, TxA2 is also capable of activating nearby platelets after its release into plasma. It has a very short half-life of 30 seconds before its conversion to the inactive metabolite thromboxane B2 prevents widespread platelet activation beyond the vicinity of thrombus formation. Both arachidonic acid and analogs of TxA2 have been found to activate and aggregate platelets by mediating shape change and phosphorylation of signaling enzymes. The thromboxane receptor (TP) is a member of the seven-transmembrane G-protein-coupled receptor family and has been localized to the plasma membrane. Two isoforms of the receptor have been identified in platelets TP𝛼 and TP𝛽 – and they activate platelets through ghe Gq pathway.

Physiologic Inhibition of Platelet Activation

One of the many remarkable features of platelets is their ability to remain in a physiologic resting state and resist becoming activated while navigating the heart, arterial, and venous circulations. Indeed, the pathologic consequences associated with widespread inappropriate platelet activation are life- and limb-threatening in the settings of well-characterized clinical disorders, such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia. The mechanisms responsible for maintaining the fine balance of keeping platelets in a resting state until they encounter a genuine need  to undergo adhesion, activation, and aggregation at the site of vascular injury are nearly as diverse as those responsible for mediating these physiologic phenomena.

Some general mechanisms involved in physiologic inhibition of platelet activation include phenomena such as a) generation of negative-regulating molecules by the platelet (e.g., cAMP), endothelium (e.g., PGI2, nitric oxide, heparan sulfate), and at distant sites (e.g., antithrombin); b) barrier of endothelial cells that prevents direct contact of circulating platelets with collagen; c) ecto-ADPase (CD39) expression by endothelial cells that metabolizes ADP secreted from platelets; d) tendency for blood flow to wash away unbound thrombin and other soluble mediators from the site of platelet plug formation; e) brief half-life of certain key platelet activators such as TxA2; f) tight regulation of the affinity state of receptors such as 𝛼IIb𝛽3; g) downregulation of signaling receptors to limit their actions; and h) inhibitory pathways mediated by ITIM-containing and/or contact-dependent adhesion receptors, such as PECAM, CECAM-1, JAM-A, and potentially others.

Receptor downregulation and desensitization

Signaling through G-protein-coupled receptors present on the surface of platelets is limited by their phosphorylation, which triggers desensitization, that is, uncoupling from G proteins, and internalization via Claritin-mediated endocytosis (for detail about G-protein coupled receptors please refers to thread "G Protein-Coupled Receptors and Second Messengers" at http://www.tomhsiung.com/wordpress/2014/09/g-protein-coupled-receptors-and-second-messengers/). G-protein kinases and 𝛽-arrestin are central to these processes. In addition, G-protein-coupled receptors interact with a myriad of other molecules that finely tune their signaling, including regulators of G-protein signaling (RGS) and GPCR-associated sorting proteins.

Inhibitory prostaglandins

Generation of the prostaglandins from arachidonic acid metabolism, such as PGI2 and PGE2 (at high concentrations), results in inhibition of platelet activation and aggregation, and counterbalances the actions of thromboxanes derived from the same pathway. While PGI2 and PGD2 inhibit platelet function at low doses, PGE2 displays a biphasic reponse, and inhibits platelet function only at higher concentrations, likely via the EP4 receptor. The inhibitory effects are mediated via G-protein-coupled receptors (IP and EP receptors, respectively) that couple to the 𝛼 subunits of Gs to regulate adenylate cyclase-mediated generation of cAMP. cAMP levels in platelets are also governed by the activity of phosphodiesterase, the enzyme responsible for cAMP metabolism. This enzyme activity is inhibited drugs such as the weak antiplatelet agent dipyridamole, the bronchodilator theophylline, and sildenafil, used to treat erectile dysfunction in men.

Nitric oxide

NO is generated by endothelial cells and platelets from L-arginine in response to shear stress forces and other platelet agonists, such as thrombin and ADP. The bulk of the evidence suggests that at high concentrations NO functions to inhibit platelet activation through the cyclic guanosine monophosphate (cGMP) second messenger generated by guanylate cyclase activation. Elevations in cGMP, by modulating phosphodiesterase activity, can raise intraplatelet cAMP. Paradoxically, low levels of NO may elicit platelet activation pathways. Endothelial NO synthase activity is enhanced during platelet activation, presumably as an additional means for limiting platelet aggregation.


Platelet Aggregation: 𝛼IIb𝛽3 (GPIIb/IIIa) Receptor and Its Signaling Mechanisms

Platelet aggregation is a complex phenomenon that is the end result of a series of adhesion- and activation-related processes. Essential components of this process include an agonist, calcium, and the adhesive proteins fibrinogen and vWF. Divalent cations, such as calcium and magnesium, are required for platelet aggregation in trace amounts, and these alter the specificity of the integrin 𝛼IIb𝛽3 for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to 𝛼IIb𝛽3, and also by the ability of the former to generate polymerized fibrin as support for the platelets in a thrombus.

The signaling pathways of 𝛼IIb𝛽3 are complex. Central concepts of the signaling pathway include inside-out signaling, which involves the processes termed affinity and avidity modulation, and outside-in signaling, in which messages are transmitted to the inside of the platelet via the events occurring outside the membrane through 𝛼IIb𝛽3 activation. Primary platelet agonists such as ADP, thrombin, and matrix proteins collagen and vWF affect platelet aggregation through a process known as inside-out signaling. In the inside-out signaling, agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails. This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, vWF, and fibronectin. In the outside-in signaling, extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tail. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.


Brief Review of Physiology of Platelet

Following injury to the blood vessel, platelets interact with collagen fibrils in the exposed subendothelium by a process (adhesion) that involves, among other events, the interaction of a plasma protein ,vWF, and a specific glycoprotein (GP) complex on the platelet surface, GP Ib-IX-V (GPIb-IX). This interaction is particularly important for platelet adhesion under conditions of high shear stress. After adherence to the vessel wall via vWF and the long GP Ib-IX-V receptor, other platelet receptors interact with proteins of the subendothelial matrix. Hereby collagen provides not only a surface for adhesion but also serves as a strong stimulus for platelet activation.

Activated platelets release the contents of their granules (secretion), including ADP and serotonin from the dense granules, which causes the recruitment of additional platelets. These additional platelets form clumps at the site of vessel injury, a process called aggregation (cohesion). Aggregation involves binding of fibrinogen to specific platelet surface receptors, a complex composed of GPIIb-IIIa (integrin 𝛼IIb𝛽3), an integrin that normally exists in a resting (low-affinity) state but that transforms into an activated (high-affinity) state when stimulated by the appropriate signal transduction cascade. GPIIb-IIIa is platelet specific and has the ability to bind vWF as well. Although resting platelets do not bind fibrinogen, platelet activation induces a conformational change in the GPIIb-IIIa complex that leads to fibrinogen binding.

Moreover, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane lipoprotein surface. During platelet activation, the negatively charged phospholipids, especially PS, become exposed on the platelet surface, and essential step for accelerating specific coagulation reactions by promoting the binding of coagulation factors involved in thrombin generation.

A number of physiologic agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 production. Other agonists, such as prostacyclin, inhibit these responses. Binding of agonists to platelet receptors initiates the production or release of several intracellular messenger molecules, including products of hydrolysis of phosphoinositide (PI) by phospholipase C, TxA2, and cyclic nucleotides. These induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and thromboxane production.