Clopidogrel attenuates coated-platelet production in patients undergoing elective coronary catheterization.

INTRODUCTION: Coated-platelets are a subclass of highly thrombotic activated platelets with an enhanced ability to generate thrombin. Excessive numbers of coated-platelets are believed to increase thrombotic risk. A previous report demonstrated that P2Y12 inhibition in vitro attenuates coated-platelet formation. The aim of this study was to determine the effect clopidogrel has on coated-platelet formation. METHODS AND RESULTS: We enrolled 27 patients undergoing elective coronary angiography. A total of 3 blood samples were taken from eligible patients: baseline, 24-hour postclopidogrel (preangiography), and 6-hour postangiography. Coated-platelet levels, expressed as percentage of total platelets, were determined with convulxin and thrombin with or without 1.5 or 6 microM adenosine diphosphate (ADP). Baseline levels of coated-platelets were 40.0% +/- 14.3% (mean +/- 1 SD). After clopidogrel exposure, the coated-platelet level was 32.8% +/- 13.6%, representing a significant 7.2% absolute reduction (AR) (17.8% relative reduction (RR); P < 0.0001). Clopidogrel significantly lowered the convulxin, thrombin plus ADP coated-platelet production (11.0% AR; 20.1% RR for 1.5 microM and 11.2% AR; 19.1% RR for 6 microM). CONCLUSIONS: This is the first report on the impact of in vivo administration of a P2Y12 antagonist on coated-platelet formation. The significance of a partial attenuation in coated-platelet potential has yet to be determined, but this could represent a new antithrombotic mechanism of clopidogrel beyond inhibition of ADP-induced aggregation.

Bax activators potentiate coated-platelet formation.

BACKGROUND: Activation of platelets with collagen plus thrombin produces a subset of cells known as coated-platelets. Coated-platelets retain several alpha-granule proteins on their surface, express phosphatidylserine (PS), lose mitochondrial potential and release microparticles. OBJECTIVE: A number of these characteristics are also observed in apoptotic cells, and this similarity leads to the hypothesis that mechanisms controlling initiation of apoptosis might also affect generation of coated-platelets. RESULTS: In this report, we demonstrate that BH3 mimetics, molecules that facilitate apoptosis by releasing pro-apoptotic Bax from its antiapoptotic partner Bcl-2, are able to promote coated-platelet formation as monitored by several different markers of these cells. Specifically, gossypol and methoxy-antimycin (MAM) promote fibrinogen retention, mitochondrial depolarization, and PS exposure upon activation with thrombin plus convulxin, a ligand of the glycoprotein VI collagen receptor. Gossypol also potentiates microparticle release by convulxin plus thrombin activated platelets although MAM does not. In addition, Bax activators together with thrombin generate coated-platelets, effectively bypassing the requirement for convulxin. CONCLUSION: These findings further support a close relationship between apoptotic-like events and the production of coated-platelets.

Density fractionation of erythrocytes by Percoll/hypaque results in only a slight enrichment for aged cells.

Density fractionation has served for many years as a standard procedure for the isolation of aged erythrocyte populations; however, a quantitative evaluation of the technique has not been available. This report describes such an analysis. Rabbits were infused intravenously with N-hydroxysuccinimido biotin to biotinylate greater than 90% of all circulating erythrocytes; the enumeration of these biotinylated cells was possible by monitoring their ability to bind avidin-coated microspheres. The biotinylated erythrocytes were shown to have a normal in vivo survival. As a result of the normal senescence process, only aged red cells would have membrane-bound biotin 50 days after biotinylation. At this time, the rabbit red cells were fractionated over Percoll/hypaque to determine the ability of density fractionation to enrich for the aged, biotinylated cells. Density fractionation resulted in an average enrichment for aged cells of only 2-3-fold above that present in a random population.

Antithrombin III does not have bound glucocerebroside.

Purified antithrombin III has been reported to have bound glucocerebroside, the major glycolipid of plasma. We have separated whole plasma by ultracentrifugation into lipoprotein-rich and lipid-deficient fractions and demonstrated that glucocerebroside and antithrombin III clearly separate into different fractions. Antithrombin III does not have glucocerebroside associated with it.

Simple and rapid purification of inside-out vesicles from human erythrocytes.

The preparation of inside-out vesicles from human erythrocytes requires their separation from contaminating right-side-out vesicles. We have taken advantage of the fact that there are no glycoproteins on the internal side of the erythrocyte membrane; therefore, inside-out vesicles do not interact with the lectin, concanavalin A, while right-side-out vesicles do interact. A concanavalin A-cellulose affinity matrix has been utilized to separate easily inside-out vesicles with a purity comparable to those prepared by prolonged centrifugation.

Studies on glutathione transport utilizing inside-out vesicles prepared from human erythrocytes.

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Lineweaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25 degrees C, this transport activity was easily lost during the storage of vesicles at 4 degrees C. The Km for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation fo transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37 degrees C, this transport activity was stable during storage of vesicles at 4 degrees C for several days. The Km for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the Km, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.

Coated-platelets: an emerging component of the procoagulant response.

Coated-platelets, formerly known as COAT-platelets, represent a subpopulation of cells observed after dual agonist stimulation of platelets with collagen and thrombin. This class of platelets retains on its surface high levels of several procoagulant proteins, including fibrinogen, von Willebrand factor, fibronectin, factor V and thrombospondin. Coated-platelets also express surface phosphatidylserine and strongly support prothrombinase activity. Retention of alpha-granule proteins on the surface of coated-platelets involves an unexpected derivatization of these proteins with serotonin and an interaction of serotonin-conjugated proteins with serotonin binding sites on fibrinogen and thrombospondin. This review will also detail experimental systems where coated-platelets are generated as a result of other agonist(s). Finally, the putative physiological consequences of coated-platelet formation will be discussed.

Quantitation of microparticles released from coated-platelets.

Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also release microparticles. Platelets were prelabeled with 2.5 microm Bodipy-maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3-0.5 microm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy-labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 +/- 5 microparticles per coated-platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated-platelet production, significantly increased the number of microparticles released per coated-platelet.

Quantitation of platelet life span in splenectomized dogs.

Platelet life spans in dogs were measured pre- and post- splenectomy utilizing in vitro whole blood biotinylation. Four splenectomized dogs were found to have significantly lengthened platelet life spans, 193 +/- 7 hours (mean +/- 1 SD;n=4) postsurgery vs. control life spans of 131 +/- 15 hours (n=6; p< 0.01) when analyzed with the multiple-hit model. Additionally, platelets from normal dogs transfused into splenectomized dogs were found to have convex survival curves with extended life spans approximating that of the splenectomized dog. These data indicate that the spleen is a significant determinant of platelet life span in dogs, with survivals increasing approximately 47% upon splenectomy.

Acute hemorrhagic complications are associated with lower coated-platelet levels in non-lacunar brain infarction.

BACKGROUND: Coated platelets are procoagulant platelets observed upon dual agonist stimulation with collagen and thrombin. Coated-platelet levels are elevated in patients with non-lacunar ischemic stroke and decreased in patients with spontaneous intracerebral hemorrhage as compared with controls. OBJECTIVE: To investigate whether acute hemorrhagic complications occurring during the initial hospital admission for non-lacunar ischemic stroke are associated with lower coated-platelet levels. PATIENTS/METHODS: Coated-platelet levels were determined in 385 consecutive patients with non-lacunar stroke. Hemorrhagic complications were defined as either intracranial hemorrhage or significant extracranial bleeding (drop in hemoglobin of ≥ 2 g dL(-1) ). The rate of acute hemorrhagic complication was compared among subjects categorized into tertiles of coated-platelet levels using an exact Cochrane-Armitage trend test. Logistic regression was used to estimate the adjusted odds of hemorrhagic complication associated with coated-platelet levels. RESULTS: Hemorrhagic complications were present in 15 (3.9%) cases. Of these, four had intracranial hemorrhage and 11 had extracranial hemorrhage. The occurrence of hemorrhagic complications differed among the coated-platelet tertiles: 10.2% for the first tertile (coated-platelet levels < 35.5%), 1.5% for the second tertile and 0% for the third tertile (coated-platelet levels ≥ 47.5%, trend test). Logistic regression showed that the odds of hemorrhagic complication in those with levels < 35.5% were 14.59 times the odds for patients with levels ≥ 35.5% (95% CI: 3.24-65.7). CONCLUSIONS: Lower levels of procoagulant platelets are associated with acute hemorrhagic complications following non-lacunar ischemic stroke. These results suggest a role for coated-platelets in risk/benefit assessment in the early stages of stroke.

Collagen response and glycoprotein VI function decline progressively as canine platelets age in vivo.

Clinical and experimental observations suggest that platelet function deteriorates quickly with cell age. However, efforts to define age-dependent alterations have detected only modest biochemical changes occurring late in the cell life span. In this report, we demonstrate two significant alterations of the collagen response occurring during in vivo aging of canine platelets: a progressive increase in the EC50 for collagen types I, III and V and the emergence of a population of aged platelets which are refractory to collagen. Experiments with convulxin, a specific agonist for the collagen receptor glycoprotein VI (GPVI), also demonstrate an age-dependent decline in activation and the appearance of a non-reactive, aged population as observed with native collagens. Our studies indicate that canine platelets have two distinct binding levels for FITC-labeled convulxin and that the higher binding level disappears upon cell aging. During these studies one dog (#428) was identified whose platelets not only failed to demonstrate an age-dependent decrease in convulxin reactivity but also maintained a high convulxin-binding ability throughout their otherwise normal life span. Transfusion of biotinylated platelets from control dogs into dog #428 showed that the expected changes in collagen response and GPVI function did not occur in the transfused platelets. These observations demonstrate that the canine platelet response towards collagen is strongly dependent upon cell-age and suggest that this functional decline is at least partly due to an extrinsic-mediated alteration, possibly proteolytic, of GPVI.

Erythropoietin potentiates thrombus development in a canine arterio-venous shunt model.

Erythropoietin (EPO) has been previously shown to affect platelet as well as red cell production. In addition, recent studies demonstrated that platelets from EPO-treated dogs are hyperreactive towards thrombin when compared to age-matched, control platelets. This report extends these observations by quantitating the thrombogenic potential of EPO in dogs. Dogs with arterio-venous (A-V) shunts received 100 U EPO/kg/day for 6 days, and thrombogenicity was serially monitored by insertion of a thrombotic surface into the A-V shunt. The resulting experimental thrombi were analyzed for platelet and erythrocyte content after formalin-fixation and chymotrypsin digestion, a technique which allows non-isotopic quantitation of cellular components. By day 5 of EPO-administration all animals demonstrated a significant increase in platelet and red cell content of the experimental thrombi; the average increase in platelet number was 2.94 +/- 0.12 fold (mean +/- 1 SE; n = 3; p = 0.006) above baseline while that for red cells was 2.46 +/- 0.18 fold above baseline (p = 0.023). After cessation of EPO, thrombogenicity returned to normal. During EPO-treatment, the percentage of thiazole orange-positive (TO+) platelets increased significantly to 17.2 +/- 1.6% (mean +/- 1 SE; n = 3) on day 5 compared to a pre-treatment level of 8.5 +/- 0.9% (p = 0.029). Although the percentage of TO+ erythrocytes also increased during the short course of EPO administration, the change was not significant. Despite the increases in TO+ cells, total platelet and erythrocyte counts did not change significantly within the time frame of these experiments. Fibrin/fibrinogen content of the experimental thrombi was unaltered with EPO-treatment. These data demonstrate that human EPO is pro-thrombotic in dogs and, in conjunction with earlier studies, suggest that hyperreactive platelets may be responsible for the potentiated thrombogenicity.

Erythropoietin administration increases production and reactivity of platelets in dogs.

Administration of erythropoietin (EPO) to adult dogs resulted in a dramatic increase in the number of thiazole orange-positive (TO+) platelets, also referred to as reticulated platelets. Pre-treatment level of TO+ platelets was 6.2 +/- 0.5% (mean +/- 1 SE: n = 5); following day 5 of treatment with 500 U EPO/kg/day, the percentage of TO+ platelets peaked at 16.8 +/- 2.3% (n = 5; p <0.02). After cessation of the hormone, the number of TO+ platelets fell rapidly to below starting levels. Unexpectedly, there was a significant decline in total platelet count during EPO administration despite an increased level of TO+ platelets. To assess platelet reactivity, total platelets and TO+ platelets from EPO-treated dogs were analyzed for thrombin-responsiveness as quantitated by P-selectin expression on the cell surface; reactivity was expressed as a thrombin EC50, the thrombin concentration required to activate 50% of platelets. Both total and TO+ platelets were hyperreactive during EPO treatment when compared either to pre-treatment values or to control animals. Thrombin EC50 values for total and TO+ platelets on day 5 fell to 66.5 +/- 5.4% (mean +/- 1 SE; n = 5; p <0.02) and 62.2 +/- 8.7% (n = 5; p <0.025), respectively, of pre-treatment levels. These data indicate that EPO not only promotes the synthesis of increased numbers of TO+ platelets in the dog but that these newly produced platelets are hyperreactive when compared to TO+ platelets from control animals.

Non-isotopic method for quantitation of platelets and erythrocytes in experimental thrombi.

Experimental animal models of thrombosis have been established in several species to examine factors responsible for thrombotic disorders in man. One technical facet of all thrombosis models is the need to quantitate cell deposition on thrombogenic surfaces, and this is routinely accomplished with radioisotopic labeling of specific components. Data reported here demonstrate that formalin-fixed thrombi can be hydrolyzed with chymotrypsin allowing recovery and quantitation of platelets and erythrocytes incorporated within the clot. Recovery of platelets from in vitro generated, model thrombi averaged 99 +/- 10% (mean +/- 1 SD; n = 7; range 88-116%) of calculated content; recovery of erythrocytes was 94.1 +/- 1.1% (n = 6) as measured by recovery of cellular hemoglobin after chymotrypsin hydrolysis of clots. Chymotrypsin was also shown to release platelets and erythrocytes from string-bound thrombi generated in vivo with an arterio-venous shunt model in beagle dogs. Platelet recovery from these string clots after chymotrypsin hydrolysis was independently verified with a quantitative Western blot assay of platelet antigens. These data demonstrate that experimental thrombi can be hydrolyzed with chymotrypsin, thereby not only eliminating the need for radioisotopes, but also permitting flow cytometric analysis of cells comprising the thrombus.

Radioisotopic assay for erythrocyte adenosine 5'-monophosphate deaminase.

Adenosine-5'-monophosphate deaminase is a critical enzyme in the regulation of adenine nucleotide levels in the erythrocyte. The routine examination of this enzyme in crude hemolysates is difficult with the commonly used assay which monitors ammonia generated by the deamination reaction. This report details a radioisotopic assay for AMP deaminase which allows separation of the [14C]inosine 5'-monophosphate product from the [14C]adenosine 5'-monophosphate substrate by ion-exchange chromatography at pH 2.2. The radioisotopic assay is linear with respect to time and enzyme concentration over a considerable range and thereby significantly simplifies the monitoring of crude or dilute enzyme preparations.

Characterization of senescent red cells from the rabbit.

The above data continue to demonstrate the metabolic well being of the aged red cell as it is isolated from rabbits. The abundance of ATP, the absence of surface-bound IgG and a variety of other observations at this time lead to the tentative conclusion that the senescent red cell is amazingly healthy. Many investigators have predicted that the red cell is removed from the circulation as a metabolically exhausted effete cell. There is currently no evidence to support this other than a decrease in deformability of the cells with time, but it is not clear that this decline in deformability is sufficient to keep the cell from circulating. In either case, many of the previously proposed causes of cellular removal are clearly incorrect for the rabbit, and it is now time to focus on new directions for observing either cellular impairment or perhaps the presence of a cellular clock which is independent of the cell's metabolic state. Another point which should be addressed is the reliability of the biotinylation model in rabbits as it relates to red cells in other species. So far several observations in aged red cells isolated with valid models have been reproduced across species boundaries including the rise in ATP, the fall in AMP deaminase activity, the shift in the 4.1a to 4.1b protein ratio, the stability of a number of glycolytic enzymes, and the instability of pyrimidine 5'-nucleotidase activity. To this point, the rabbit has been a reliable model of red cell aging and one with implications for other species.

Coated-platelet levels and progression from mild cognitive impairment to Alzheimer disease.

OBJECTIVES: Coated-platelets are a subset of platelets produced by dual-agonist activation with collagen and thrombin. These platelets retain full-length amyloid precursor protein on their surface, are elevated in patients with amnestic as compared to nonamnestic mild cognitive impairment (MCI), and correlate with disease progression in Alzheimer disease (AD). Prompted by these findings, we investigated the association between coated-platelet production in amnestic MCI and rate of progression to AD. METHODS: Coated-platelet levels were assayed in 74 patients with amnestic MCI who were subsequently followed longitudinally for up to 36 months in an outpatient dementia clinic. Levels are reported as percent of cells converted into coated-platelets. Subjects were categorized into tertiles of coated-platelet levels. The distributions of time to progression to AD were estimated for each tertile using cumulative incidence curves and compared statistically using a log-rank test. Cox proportional hazards regression was used to adjust for potential confounders. RESULTS: The 24-month cumulative incidence of progression to AD was different among tertiles: 4% for the first tertile (lowest coated-platelet levels), 13% for the second tertile, and 37% for the third tertile (overall log-rank test, p = 0.02). The hazard rate of progression to AD for patients in the highest coated-platelet tertile was 5.1 times that for patients in the lowest tertile (p = 0.04), whereas the hazard rate for the middle tertile was similar to that for the lowest tertile (hazard rate ratio = 1.5, p = 0.7). CONCLUSIONS: Elevated coated-platelet levels in patients with amnestic MCI are associated with increased risk for progression to AD.