The platelet proaggregating and potentiating effects of unfractionated heparin,low molecular weight heparin and heparinoid in intensive care patients and healthy controls

Abstract: Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose-dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.     

Effects of Cod Liver Oil on Platelets and Coagulation in Familial Hypercholesterolemia (Type IIa)

Patients with familial hypercholesterolemia (type IIa) were given 30 ml cod liver oil (CLO) as dietary supplement daily for 6 weeks. The effects on platelets, bleeding time, coagulation and blood and platelet lipids were examined. The major findings were a reduced collagen-induced platelet aggregation and a decrease in thrombin-stimulated thromboxane B₂ generation in platelets in vitro. The primary bleeding time was not significantly prolonged. Statistically significant increase in ***eicosapentaenoic add/arachidonic acid ratios in the main platelet phospholipids were also observed. These changes did not correlate with any of the changes in platelet behavior observed after CLO intake. The serum total and HDL cholesterol and triglycerides were not altered during the trial.     

In vitro and in vivo effects of desmopressin on platelet function.

BACKGROUND AND OBJECTIVE: Desmopressin (DDAVP) may shorten bleeding time in patients with disorders of platelet function, but its mechanism of action in these conditions is still a matter of debate. In particular, contrasting results have been obtained concerning the ability of DDAVP to interact with platelets and to activate them directly. To gain further information on the DDAVP-platelet interaction, we studied the in vitro and ex vivo effects of DDAVP on platelet function. DESIGN AND METHODS: Platelet responses to DDAVP both as a single agent and in conjunction with agonists of platelet activation were investigated. For in vitro experiments platelets were obtained from healthy adult volunteers, while the ex vivo effects of DDAVP were studied in 12 patients with a bleeding disorder receiving a test dose of this drug. RESULTS: DDAVP in vitro did not induce either platelet aggregation or surface expression of the activation-dependent antigens; it did, however, greatly inhibit platelet aggregation response to vasopressin (AVP) and increased the maximal extent of platelet aggregation induced by collagen and ADP. DDAVP infusion did not promote the expression of activation antigens, but significantly enhanced ex vivo platelet aggregation stimulated by ADP and collagen. This priming effect was observed in patients with von Willebrand's disease, hemophilia A, May-Hegglin anomaly, gray platelet syndrome and Ehlers-Danlos syndrome. In all these patients bleeding time was shortened by DDAVP infusion. In contrast, neither platelet aggregation nor bleeding time was modified in two subjects with Glanzmann's thrombasthenia. INTERPRETATION AND CONCLUSIONS: Our in vitro experiments indicate that DDAVP interacts directly with platelets and facilitates their activation via other agonists. In vivo results suggest that this effect occurs and is clinically relevant in patients with platelet dysfunction responding to DDAVP with a shortening of bleeding time.     

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4)

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.     

Further evidence that the residual vWf:Ag in porcine FVIII:C induces human platelet aggregation

Porcine or bovine factor VIII concentrates (FVIII:C) have been used during the past 3 decades to control bleeding in patients who have developed antibodies to human factor VIII. Since current preparations of animal FVIII:C are not known to transmit infectious agents such as hepatitis or human immunodeficiency virus, they are of potential therapeutic interest. A purified porcine FVIII:C (Hyate:C) is now widely used as an alternative to human FVIII:C in patients with inhibitor. Unlike earlier preparations of porcine FVIII:C, thrombocytopaenia is rare with the current preparation. Nonetheless, it causes the aggregation of human platelets in vitro. Our aim was to identify precisely the plasma factor which induces platelet aggregation. The effects of commercial porcine FVIII:C, porcine fibrinogen, porcine fibronectin and the corresponding preparations from human origin on platelet aggregation were studied. Platelet aggregation was quantified by measuring the fall in single platelet count in human whole blood. Of these preparations, only porcine FVIII:C (0.1-1 U/ml) and porcine fibrinogen (80-600 micrograms/ml) induced a fall in single platelet count of up to 85% due to aggregation. The extent of aggregation was directly proportional to the amount (0.007-0.1 U/ml test aliquot) of residual von Willebrand factor antigen (vWf:Ag) in the preparations. A monoclonal antibody to vWf:Ag inhibited the aggregation. We believe that the aggregation of human platelets induced in vitro by porcine FVIII:C is mediated by vWf:Ag which also may be responsible for thrombocytopaenia reported following administration of porcine FVIII:C in vivo.     

Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: studies on one hundred six patients

One hundred six patients with storage pool deficiency (SPD) were studied with respect to platelet count, bleeding time, total platelet ATP and ADP, platelet serotonin, and in vitro aggregation. The diagnosis of SPD was made on basis of a prolonged bleeding time, a decreased total platelet ADP, and a diminished level of serotonin. Fifty-one patients from 34 unrelated families had congenital SPD, and 55 patients had acquired SPD. Congenital SPD was a common disorder in patients with a lifelong bleeding tendency and a prolonged bleeding time. The frequency in this group of patients was 18%, about one-half the frequency of von Willebrand's disease (vWd). Twenty-three percent of all patients had normal aggregation responses to ADP, epinephrine, and collagen; 33% had aggregation tracings typical for a secretion defect; and 44% had miscellaneous aggregation abnormalities. These findings indicate that SPD is common, heterogeneous, and not necessarily associated with in vitro aggregation abnormalities.     

Bernard-Soulier Disease: a Study of Four Patients and their Parents

S ummary. Two families with Bernard-Soulier disease, including four patients and three of their parents, were studied and detailed clinical summaries are presented. One patient in each family has suffered severe bleeding problems while the other affected sibling is less severely affected. There has been no excessive bleeding in any of the parents or other family members. The patients demonstrated the abnormalities characteristic for Bernard-Soulier disease: thrombocytopenia, giant platelets, prolonged bleeding time, abnormal platelet aggregation to human FVIII_vWF and ristocetin or bovine FVII_vWF alone, defective ristocetin-induced binding of human ¹²⁵I–FVIII_vWF multimers, decreased platelet lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. The parents had normal platelet counts, bleeding times, and FVIII-mediated aggregation. However, the parents had anormally large platelets, decreased sensitivity to lysis by a drug-dependent antibody and complement, and a decreased concentration of membrane glycoprotein I. Therefore the heterozygous state for Bernard-Soulier disease is recognizable by platelet membrane abnormalities although there is no defect of platelet function and no excessive bleeding. Red cell membrane proteins of one patient were normal, suggesting that phenotypic expression of the Bernard-Soulier disease defect is restricted to platelets.     

Effect of alternate-day regular and enteric-coated aspirin on platelet aggregation, bleeding time, and thromboxane A2 levels in bleeding-time blood

The effectiveness of low-dose aspirin for primary prevention of cardiovascular mortality is being assessed among the nearly 22,000 United States physicians currently participating in the Physicians' Health Study. Because of occasional reports of gastric irritation among study participants, two enteric-coated aspirin preparations were tested as possible alternatives to regular compressed aspirin for platelet inhibition. Thirty-three volunteers were assigned randomly to one of four treatment groups: regular aspirin (325 mg), placebo, and two enteric-coated aspirin preparations (325 mg). Pills were administered every other day, duplicating the regimen used in the Physicians' Health Study. Bleeding times, platelet aggregation, and thromboxane A2 levels produced by aggregating platelets in vitro, as well as in collected bleeding-time blood, were determined. Measurements were taken before and after a single dose as well as after seven alternate-day doses. Regular and enteric-coated aspirin preparations were equally efficacious in prolonging the bleeding time, inhibiting platelet aggregation, and suppressing thromboxane A2 production. There was virtually complete suppression of thromboxane A2 production (over 99 percent), by platelets in vitro and in collected bleeding-time blood. The levels were still profoundly reduced (89 percent) 48 hours after the last dose. Enteric-coated aspirin may provide an alternative to regular aspirin in a low-dose regimen designed to inhibit platelet activity.     

Heparin-associated thrombocytopenia: Successful therapy with the heparinoid Org 10172 in a patient showing cross-reaction to LMW heparins

Summary A patient suffering from heparin-associated thrombocytopenia (HAT), recurrent arteriothromboses, and acute renal failure after treatment with standard heparin is described. He failed to improve when therapy was continued with low-molecular-weight (LMW) heparin (Fragmin, Kabi Pfrimmer, Erlangen, FRG). By means of the in vitro heparin-induced platelet activation (HIPA) assay it was shown that standard heparin and the LMW heparins Fragmin and Fraxiparin (Sanofi Labaz, Munich, FRG), as well as the enoxaparine Clexane (Nattermann, Cologne, FRG), all induced platelet activation with the patient's serum. In contrast, the LMW heparinoid Org 10172 (Organon, Oss, The Netherlands) did not cause platelet activation. When the patient was subsequently treated by parenteral administration of Org 10172 as anticoagulant over a period of several weeks the number of platelets rapidly increased and the patient almost completely recovered. This case shows that strong in vivo and in vitro cross-reactivity between standard heparin and LMW heparins may occur, but can be avoided by the use of a novel heparinoid, Org 10172. The HIPA assay provides a simple and sensitive laboratory method for the choice of an innocuous heparin or heparinoid for continued parenteral anticoagulation.     

An atypical von Willebrand's disease with hyperreactivity of platelet aggregation

Two family members (daughter and mother) with a bleeding disorder showed prolonged bleeding time and activated partial thromboplastin time associated with decreased plasma levels of factor VIII procoagulant activity, factor VIII-related antigen, and factor VIII-ristocetin cofactor activity. The ristocetin-induced platelet agglutination (RIPA) was enhanced, ADP-, collagen- and Ca ionophore-induced platelet aggregation were also increased at low concentrations of these compounds. In the mother, spontaneous platelet aggregation (SPA) was also observed. Contrary to type II B von Willebrand's disease (vWd), pseudo-vWd and platelet-type vWd, the patients did not show any increased binding of factor VIII/vWf to platelets in the presence of ristocetin. The RIPA in normal controls were inhibited by addition of antifactor VIII antiserum to the platelet-rich plasma, but not in cases 1 and 2. In this atypical vWd, therefore, the hyperreactivity of platelet aggregation may be due to an intrinsic abnormality of the platelets, but not to any enhanced interaction between plasma factor VIII and the platelets.     

The effect of somatostatin on platelets: in vivo and in vitro studies

The influence of somatostatin on platelets was studied in healthy volunteers. After a bolus injection of 250 microgram somatostatin followed by a three hour infusion at a rate of 250 microgram somatostatin/hr a statistically significant fall of platelet count and impairment of platelet aggregation was observed. The aggregation inhibiting effect of somatostatin at the end of the three hour infusion is clinically unimportant as compared to the effect of aspirin. In vitro concentrations up to 10.0 microgram somatostatin per ml do not show any effect on platelet aggregation and platelet stickiness. Endocrinologically active doses of somatostatin in short term infusions are very unlikely to cause bleeding disorders.     

Essential athrombia: study of a new case.

A constitutional platelet function disorder in a twelve year-old girl characterized by a lifelong bleeding tendency, prolonged bleeding time, normal platelet count, normal clot retraction, normal platelet factor 3 activity and impaired platelet aggregation was reported. Platelet aggregation, studied turbidimetrically, was absent in the presence of usual doses of ADP (1-4 MUM) although a small wave of primary aggregation was obtained by very large ADP concentrations (25-50 muM). The platelets were also unresponsive to epinephrine, thrombin and diluted collagen suspensions. But an almost normal aggregation response occurred with strong collagen suspensions. The platelets responded to Ristocetin. Release of platelet ADP was found to be normal by collagen and thrombin, but impaired by kaolin. Platelet fibrinogen content was normal. The present case, investigated with recent methods, confirms the existence of a type of primary functional platelet disorder characterized solely by an aggregation defect, described in 1955 and 1962 under the name of "essential athrombia."     

Partial inhibition of platelet aggregation by nebulized pentamidine in severe haemophiliacs

The antiparasite agent pentamidine has been shown to inhibit human platelet aggregation in vitro at concentrations that (potentially) may be attained in patient plasma after the administration of the drug by nebulizer. We measured platelet aggregation in platelet-rich plasma (PRP) before and after the administration of 300 mg nebulized pentamidine to 10 HIV-positive patients with severe haemophilia on prophylaxis against Pneumocystis carinii pneumonia. All patients had normal platelet counts. PAF-acether, U46619, collagen and ADP at different concentrations were used as agonists. Platelet aggregation was lower in PRP samples taken at the end of pentamidine administration and 1 h thereafter than in samples taken at the same time points in control experiments (without the administration of pentamidine). The inhibition of platelet aggregation was mild and tended to be overcome by higher concentrations of platelet agonists. The bleeding time was prolonged from 5 to 15 min in one patient but did not change in the remaining nine patients. In conclusion, this controlled study shows that nebulized pentamidine inhibits platelet aggregation in HIV-positive haemophiliacs without significantly affecting their bleeding times. Although this mild inhibitory effect may not be clinically relevant in haemophiliacs with normal platelet counts despite their defect in intrinsic coagulation, patients with HIV-related thrombocytopenia should be monitored to detect any excessive prolongation of their bleeding times after nebulized pentamidine.     

Augmentation by platelets of granulocyte aggregation in response to chemotaxins: studies utilizing an improved cell preparation technique

Considerable evidence exists to suggest roles for both platelets and granulocytes (PMNs) in pulmonary injury in shock. While believing that the major contributions of the two cell types are sequential, in vitro observations suggested that direct interactions between granulocytes and platelets might also amplify tissue damage. Using isotonic Percoll density gradients to isolate PMNs, we therefore studied the effect of deliberate platelet contamination on PMN aggregation. PMN aggregation in response to N-formyl-met-leu-phe or activated complement was enhanced by the presence of 1 platelet/PMN, an effect that became maximal at 16 platelets/PMN (p less than 0.01); large mixed aggregates were formed. Lysed, aspirinated, and indomethacin-treated platelets retained their augmentative capacity, as did platelets washed by gel filtration. The effect was not mimicked by the addition of histamine or serotonin to PMN preparations. None of these platelet preparations augmented lysosomal enzyme release. We conclude that platelets augment PMN aggregation, both by forming giant mixed PMN/platelet aggregates and also by producing a labile augmentative substance, the production of which may be independent of thromboxane synthesis. We propose that direct as well as sequential platelet/PMN interactions may be important in tissue injury in shock.     

Effect of ketorolac and low-molecular-weight heparin individually and in combination on haemostasis.

Low-molecular-weight heparins, when used in surgical patients for thromboprophylaxis, may be used concurrently with ketorolac, a non-steroidal anti-inflammatory drug that is used for analgesia. Because these two agents can influence the haemostatic system, it is important to identify any such effect. The haemostatic interaction between dalteparin and ketorolac was assessed in a double-blind, placebo-controlled, randomized, crossover study of healthy male volunteers each given all four combinations of ketorolac/placebo and dalteparin/placebo. The effect of ketorolac and dalteparin on haemostasis was assessed by measuring in-vitro platelet aggregation, anti-factor-Xa, activated partial thromboplastin times and skin bleeding time. The results were analysed for evidence of an interaction between ketorolac and dalteparin. Ketorolac inhibited platelet aggregation in whole blood and platelet-rich plasma. The administration of dalteparin led to a significant increase in levels of anti-factor-Xa and a significant prolongation in the activated partial thromboplastin time, although it remained within the range of the normal population. There was no evidence of any interaction between ketorolac and dalteparin with regard to platelet aggregation, anti-factor-Xa activity or activated partial thromboplastin time. The administration of ketorolac significantly prolonged the skin bleeding time. There was a significant interaction between ketorolac and dalteparin to prolong the bleeding time, although dalteparin alone had no effect on bleeding time. There was an interaction between ketorolac and dalteparin, which affected bleeding times. Such an interaction raises the possibility of haemorrhagic complications developing perioperatively when these agents are used concomitantly. Further studies are required to examine the clinical importance of this interaction.     

Modulatory effects of Escherichia coli capsular-derived sulfaminoheparosans and heparins on tissue factor-mediated activation of platelets: flow cytometric analysis.

Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.     

Reduced thiols and the effect of intravenous nitroglycerin on platelet aggregation

Nitroglycerin inhibits platelet aggregation in vitro and this effect may be important in its overall mechanism of action. In addition, its use has been associated with prolonged bleeding times and hemorrhagic complications. Despite these experimental and clinical observations, no significant antiplatelet effect of nitroglycerin has been observed ex vivo during intravenous nitroglycerin administration to patients. Because the in vitro antiplatelet effects of nitroglycerin have been shown by one of the investigators participating in this study to depend on the presence of sufficient stores of reduced intracellular thiol--which are readily depleted ex vivo by nitroglycerin in the formation of S-nitrosothiols--an attempt was made to unmask nitroglycerin-mediated inhibition of platelet aggregation by exposing platelets taken from patients treated with nitroglycerin to the reduced thiol N-acetylcysteine ex vivo. The obtained data demonstrate that platelets taken from patients treated with intravenous nitroglycerin manifest attenuated aggregation responses ex vivo when thiol stores are repleted. It is therefore proposed that the mechanism of action of nitroglycerin is mediated in part by its antiplatelet effect and that this effect depends on the adequacy of reduced intracellular thiol stores.     

Comparison of the platelet pro-aggregatory effect of conventional unfractionated heparins and a low molecular weight heparin fraction (CY 222)

Two unfractionated heparins (UH), a porcine intestinal mucosal heparin (PIM), a bovine lung heparin (BLH) and a low molecular weight heparin (LMWH), CY 222, were assessed for their capacity to enhance platelet aggregation in vitro. Their potential proaggregatory effect was investigated in four systems: (a) enhancement of submaximal platelet aggregation induced by conventional agonists in platelet rich plasma and (b) in whole blood; (c) reversal of inhibition of platelet aggregation induced by iloprost, a stable analogue of prostacyclin; (d) the direct aggregatory effect of these anticoagulants on hyperactive platelets prepared from patients with severe peripheral vascular disease or anorexia nervosa. Whereas BLH and PIM, at therapeutic concentrations, had a proaggregatory effect in all four systems, CY 222 had no significant effect when compared with the controls. BLH was more potent than PIM in three of the four systems studied. These observations confirm that conventional UH are more proaggregatory than LMWH, and thus the latter may be potentially safer. These observations are also consistent with the fact that BLH administration causes thrombocytopenia more frequently than PIM. The direct activation by UH of platelets from patients not previously exposed to heparin also challenges the hypothesis that heparin-induced platelet activation and thrombocytopenia is solely mediated by classical heparin-dependent immune mechanisms.     

Haemorrhagic Thrombocytopathy Associated with Dilatation of the Platelet—Membrane Complex

We evaluated eight patients from four families because of a history of excessive bleeding. Most patients had prolonged bleeding times, absent secondary wave of platelet aggregation in response to epinephrine, collagen and adenosine diphosphate (ADP), and defective ¹⁴C-serotonin release and platelet factor 3 availability. These findings are characteristic of a platelet release defect. Electron-microscopic examination of the platelets of seven patients revealed a common abnormality. From 30% to 70% of the platelets in any given sample exhibited a prominent membrane complex and dilated, tortuous surface-connected canalicular system ('swiss-cheese' platelet). In two patients there was coincident storage-pool disease, but the remainder had adequate dense bodies and a normal ratio of ATP to ADP. SDS-polyacrylamide gel electrophoresis of platelet proteins and glycoproteins showed no abnormalities. The patency of the canalicular system was demonstrated in one patient by the observation that dense bodies appeared in the cannaliculi and outside the platelets following collageninduced aggregation of polylysine-treated platelets. Since platelet-aggregation responses to the calcium ionophore A23187 were normal, we conclude that the defective platelet function in these patients may be due to impaired calcium mobilization from the morphologically abnormal membrane complex.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.