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In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF-induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.

Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet-rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

To assess the nature of the hemostatic abnormalities associated with chronic liver disease, we have simultaneously evaluated the kinetic of radiolabeled platelets, fibrinogen, and plasminogen, together wit tests of platelet and fibrinogen function, and coagulation factors in 60 patients with documented, severe but stable cirrhosis of the liver. The mean platelet survival was substantially reduced (5.8 +/- 1.7 days compared with 9.5 +/- 0.6 days in normals, p less than 0.0001) and splenic sequestration of platelets was increased (mean recovery was 47% +/- 18 vs. 65% +/- 5 in normals, p less than 0.0001). Nevertheless the mean platelet count was nearly normal because platelet production was increased nearly twice normal values (mean turnover was 64,000 +/- 33,000 platelets/microliter/day; p less than 0.0001). The mean rate of fibrinogen removal was shortened (p less than 0.0001) and fibrinogen turnover increased about 20% (p = 0.008) while the mean fibrinogen concentration was not different from the results in normal control subjects (p = 0.212). Autologous and homologous fibrinogen disappeared from the circulation at equivalent rates (r = 0.751; p = 0.008), indicating that fibrinogen from cirrhotic patients was not kinetically different from normal fibrinogen. The mean plasminogen survival was significantly shortened (p less than 0.0001), but the mean plasminogen turnover was not increased (p = 0.388). Thus the plasminogen concentration was reduced (p less than 0.0001). For platelets, fibrinogen, and plasminogen, the production rate was the most important determinant of the concentration in the circulation. The administration of heparin to cirrhotic patients improved the survival of fibrinogen but not of platelets. LeVeen valve implantation generally resulted in parallel shortening of both the platelet and fibrinogen survivals and concentrations. Platelet function as assessed by template bleeding time, platelet retention by glass bead columns, and aggregation induced by ADP, epinephrine, and collagen was normal. Fibrinogen determinations by the Clauss and Jacobsson techniques were equivalent, indicating that the ability to polymerize fibrin monomer was not detectably altered. Sixty percent of patients had an abnormal prothrombin time and half that number had a prolonged partial thromboplastin time. Although most patients had a modest decrease in the prothrombin complex coagulation factors, fibrin degradation products were, in general, not elevated in the circulation. The wide range of values observed suggests that a number of different and complex processes may be ongoing in different patients. Overall, the kinetic data suggest that platelets are initially consumed, perhaps on incompletely endothelialized endovascular surfaces in the liver, and that fibrin subsequently forms secondary to local stasis. The absence of increased production of fibrinogen and plasminogen despite shortened survival times reflects the reduced capability of the cirrhotic liver to increase protein synthesis.

Changes in circulating platelets were observed after placement of woven Dacron aortic prostheses in dogs. Platelets had increased sensitivity to aggregating agents for up to 6 months postoperatively, during which period platelet survival was maximally shortened. Platelet survival gradually lengthened but remained less than preoperative values, even at 18 months. Platelet 5HT, consistently reduced after placement of aortic prostheses, closely paralleled changes in platelet survival. Studies with platelets labeled simultaneously with 14C-5HT and 51Cr suggested in vivo platelet release and reutilization of 5HT. In vitro platelet uptake of 14C-5HT, however, was depressed. Urinary excretion of 5HIAA increased slightly, but not significantly, after placement of aortic prostheses. These findings suggested that platelet interaction with the prosthetic surface led to partial release of platelet storage granule contents. Reutilization of released 5HT occurred but was limited because of impaired platelet uptake or storage of 5HT. Platelet damage was suggested by 51Cr-platelet cross-transfusion experiments. Platelets from normal dogs had normal recovery and reduced survival when cross-transfused into animals with aortic prostheses. When platelets from dogs with aortic prostheses were cross-transfused into normal animals, recovery was strikingly reduced and survival very short. Taken together, these studies suggest that, in dogs, platelet interaction in thrombosis on prosthetic surfaces is, in part, a reversible process, with platelets re-entering the circulation in an altered state.

Thrombo-embolic events remain one of the most serious complications in patients with nephrotic syndrome (NS). A causal relationship between coagulation abnormalities and the occurrence of thrombosis in NS has been searched. Several studies have demonstrated multiples abnormalities in the coagulation factors and platelets; however those studies did not provide adequate explanations for the thrombotic tendency of NS. This thrombotic tendency could be related to deficiency in natural coagulation inhibitors. Plasma ATIII level has been reported to be decreased both in some adults and children. Protein C is found normal or high in NS whereas protein S is increased. Nevertheless a possible decrease in functional protein S cannot be excluded.

A patient with hairy cell leukemia developed purpura not attributable to thrombocytopenia. We found markedly reduced platelet aggregation responses and malondialdehyde production, decreased serotonin uptake, and depleted dense granule contents. Ultrastructural studies showed that most platelets had few or nor granules. All of the clinical and laboratory studies of platelet function and morphology improved after splenectomy. These findings indicate that qualitative defects in platelet function occurring in hairy cell leukemia may cause clinically important bleeding and that the bleeding diathesis may be ameliorated by splenectomy.

Spontaneously hypertensive rats (Okamoto-Wistar) as compared to their normotensive controls (Kyoto-Wistar) presented a markedly higher platelet activity both in coagulation (as evaluated by the recalcification plasma clotting time of platelet-rich plasma) and aggregation, as triggered by thrombin. By comparing animals fed saturated or polyunsaturated fat, it could be observed that the saturated fat diet induced similar results on platelet functions to these observed in hypertension. In addition, the saturated fat diet further increased the platelet response of the hypertensive animals. By contrast, the polyunsaturated fat, could neither reduce the hypertension nor completely abolish the platelet reactivity associated with the hypertension. The most significant change induced by the saturated fat diet in the fatty acid composition of the platelet phospholipids was an increase in 20:3 omega 9, further enhanced in the hypertensive animals. In the polyunsaturated diet-fed rats, it was mostly 20:4 which was more elevated in the platelet phospholipids of the hypertensive. As a result, it was the sum of 20:3 omega 9 + 20:4 in the platelet phospholipids, which appeared to be the most significantly related, in the 4 groups of animals, to the response of platelets to thrombin induced aggregation.

A diminished chemotactic response was observed with the neutrophils of a patient with the Chediak-Higashi syndrome, who was not in the accelerated phase of the disease. An abnormally low release of myeloperoxidase from these cells during phagocytosis was also noted; this resulted in a decreased iodination capacity and probably also caused the defect in the intracellular killing of bacteria by the neutrophils. The level of cyclic AMP in these cells was elevated, but decreased after treatment with ascorbate either in vitro or in vivo. During ascorbate therapy, the bactericidal activity of the neutrophils normalized, whereas the chemotactic response remained low. Nevertheless, the patient had significantly less infections during ascorbate therapy. The bleeding tendency, due to a storage-pool disorder of the Chediak-Higashi platelets, was unaffected by treatment with ascorbate. The patient's lymphocytes did not display any activity in antibody-dependent lymphocytotoxicity. This defect was not affected by treatment with ascorbate either.

Prolonged mean template bleeding time of 14 min observed in seven cattle with the Chediak-Higashi syndrome (CHS) prompted the examination of platelet function in these animals. There was no distinguishable difference in concentration of circulating platelets between CHS and control cattle. CHS bovine platelets failed to aggregate in vitro in response to concentrations of acid-soluble collagen which induced aggregation in all normal samples. The primary platelet response to 5 muM ADP was normal in CHS cattle. A markedly decreased amount of serotonin (1.2% of normal) was detected in CHS bovine platelets. Bovine CHS platelet ATP and ADP contents were significantly less than normal, and the ATP/ADP ratios were 5.04 in normal and 29.38 in CHS platelets. Results of these animal investigations prompted a similar study of two patients with CHS. In humans, an increased bleeding time greater than 15 min and an in vitro impaired aggregation response to acid-soluble collagen and 5 muM adrenaline were discovered. Both ATP and ADP were reduced in CHS human platelets, and the ATP/ADP ratio was 3.96, compared to a ratio of 1.52 for platelets of two normal subjects. These findings suggested the presence of a "storage pool disease" of CHS platelets.

Various defects in platelet function have been reported as being associated with multiple myeloma. In 30 myeloma patients and 15 healthy controls, we investigated platelet survival using in vitro labeling of autologous platelets with 111indium-oxine and measuring the in vivo kinetics of the radioisotope. Significantly shortened platelet half-life in patients averaged 73 hours, while platelet half-life in the healthy controls averaged 107 hours. In myeloma patients, serum levels of thromboxane B2, beta-thromboglobulin, and platelet factor 4 were significantly elevated; aggregation indices were within the pathological range; platelet counts and spleen-liver indices, however, were comparable to those of the healthy control group. No statistical correlation was found between platelet half-life and paraprotein concentrations. Our findings suggest an initial--so far unexplained--intravascular process of platelet activation and consumption that finally manifests in shortened platelet half-life. It seems that overt thrombocytopenia develops only when the compensatory capacity of the bone marrow finally becomes exhausted. Further studies should be able to elucidate the pathophysiologic processes involved.

The deformability of human platelets has been evaluated by micropipette aspiration. Control discoid platelets were about ten times as resistant to deformation in the micropipette as red blood cells. Under a constant negative pressure of 10 cm H2O, control platelets developed extension lengths of 0.74 +/- 0.1 micron. Prior treatment with vincristine, colchicine, or low temperature, all of which remove platelet microtubules, was associated with marked increases in lengths of aspirated segments. Taxol or heavy water, which stabilize microtubules, prevented the increased deformability caused by agents that dissolve microtubules. Cytochalasin B, an agent that inhibits assembly of actin microfilaments, also caused an increase in lengths of aspirated segments that could not be prevented by taxol. Vincristine and cytochalasin B, together, caused a greater increase in deformability than either agent alone. These results indicate important roles for microtubules and microfilaments in platelet deformability.

Platelet function studies were performed on two patients with the Chediak-Higashi syndrome, one of whom had a history of easy bruising unrelated to thrombocytopenia. Both patients had prolonged bleeding times, abnormal platelet aggregation, and a defect of platelet storage granules, manifested by reduced platelet ADP, an increased ATP/ADP ratio, increased adenine nucleotide specific radioactivity after 3H-adenine labeling, and decreased platelet uptake of radioactive 5-hydroxytryptamine. These findings confirm preliminary data in animals with the Chediak-Higashi syndrome, provide and explanation for impaired primary hemostasis in these patients, and illustrate another disorder in which platelet storage-pool deficiency occurs.

To elucidate the usefulness of beta-thromboglobulin (beta TG) in the differentiation of the mechanism of thrombocytopenia, plasma beta TG concentration was measured in one patient with amegakaryocytic thrombocytopenia, four patients with autoimmune thrombocytopenia (ATP), two patients with thrombotic thrombocytopenia (TTP), and one patient with thrombocytopenia secondary to disseminated intravascular coagulation (DIC). Plasma beta TG was not measurable in amegakaryocytic thrombocytopenia, was normal in ATP, and was increased in TTP and DIC. These data indicate that in thrombocytopenic patients, increased plasma beta TG concentration may result from intravascular platelet consumption with release of platelet constituents in contrast to extravascular platelet destruction by the macrophage-monocyte system.

Platelet activation was assessed in hospitalized third-trimester patients with preeclampsia (n = 11) or chronic hypertension with superimposed preeclampsia (n = 11) and in healthy outpatient pregnant controls (n = 10) by measuring plasma beta-thromboglobulin, platelet factor 4, the platelet aggregate ratio, and the amount of collagen required to produce half-maximal aggregation velocity (Kd). Only plasma beta-thromboglobulin levels differed significantly between patients with preeclampsia (50.1 +/- 37.9; p less than 0.05) or chronic hypertension with superimposed preeclampsia (47.6 +/- 16.3; p less than 0.01) and the control subjects (22.5 +/- 11.3). beta-Thromboglobulin values in patients with preeclampsia, but not chronic hypertension with superimposed preeclampsia, correlated directly with 24-hour urinary protein loss (r = 0.93, p less than 0.001) and serum creatinine levels (r = 0.62, p less than 0.05) and inversely with creatinine clearance (r = 0.60, p = 0.05). We conclude that (1) beta-thromboglobulin is elevated in patients with preeclampsia or chronic hypertension with superimposed preeclampsia, (2) the normal platelet aggregate ratio and the Kd indicate that the increase in beta-thromboglobulin is not due to an intrinsic change in platelet responsiveness, and (3) the elevation of beta-thromboglobulin in patients with either preeclampsia or chronic hypertension with superimposed preeclampsia appears to be secondary to platelet consumption in the microvasculature, although in patients with preeclampsia altered renal function may be contributory.

An experimental preparation was developed to assess changes in surface-induced arterial thrombosis in nine dogs with woven Dacron aortic prostheses. The preparation consisted in measuring the occlusion time of an A-V shunt with controlled flow rate (200 mg/min). This measurement depended on formation of an occlusive, platelet-rich thrombus on a diaphragm of polyester mesh. Two to 6 months after placement of thoracoabdominal aortic prostheses, all animals demonstrated striking prolongation of A-V shung occlusion time. This occurred in the presence and absence of heparin anticoagulation. No change in A-V shunt occlusion time was noted in dogs who had sham operations. These findings complement out studies showing biochemical and functional changes in circulating platelets in dogs with aortic prostheses, and together they are consistent with mild platelet damage sufficient to impair surface-induced thrombosis. They also support our contention than, in dogs, platelets interact reversibly with prosthetic surfaces.

In 23 patients with untreated idiopathic thrombocytopenic purpura (TP), the relation of thrombokinetics to quantitative determinations of megakaryocytes in bone marrow sections was studied. The megakaryocytes were classified into maturation stages, and platelet sizes were determined. Megarkaryocyte number and volume per microliter of bone marrow were significantly higher in ITP as compared to controls. The megakaryocyte number and volume were inversely related to the peripheral platelet count. Platelet production rate was significantly increased in ITP and related to the megakaryocyte number and volume. The megakaryocytes were shifted towards more immature forms in ITP, suggesting an increased turnover rate of the expanded recognizable metakaryocyte compartment. Platelet size was significantly increased in ITP, and the mean platelet diameter was 1.6 times normal. There was a significant relationship between platelet size and platelet production rate, as well as an inverse relationship between platelet size and platelet mean life-span (MLS). There was also a significant correlation between platelet size and the proportion of young megakaryocytes.

It is suggested that an occlusive thrombus in a coronary artery develops at the site of an intimal tear but that in the interval between initiation of the thrombus and occlusion of the artery emboli are likely to be shed continually, causing multiple occlusions of myocardial vessels in the area of supply. Multiple and confluent areas of ischaemia can progress to complete occlusion of the coronary artery, or the intimal tear can regress and heal. Such a pathogenesis could account for the very variable clinical and pathological findings in myocardial infarction and for the apparently paradoxical time-relationship between coronary artery thrombosis and myocardial infarction.

Accumulation of arterial platelets was calculated from 51Cr radioactivity in the intima-inner media of flushed, perfuse-fixed aortas and branch vessels of cynomolgus monkeys 48 hours after labeling of the blood platelets. In normo-cholesterolemic controls the radioactivity per square centimeter of tissue was consistently higher in aortic branching sites (circumostial aorta) than in the remainder of the aorta. In monkeys given 10 and 100 days of hypercholesterolemic diet radioactivity rose in circumostial aorta in proportion to increases in streaks and in areas of Evans blue uptake. In branch artery inner wall, where intimal changes were minimal and usually absent, counts were significantly greater in animals given 100 days of hypercholesterolemic diet than in controls. After 10 days of hypercholesterolemic diet the mean radioactivity in aortic branch artery inner wall uniformly exceeded control values, but usually nonsignificantly, permitting the speculation that changes in platelet-intima interactions may occur in widespread fashion throughout the arterial tree very early in hypercholesterolemia when lesion formation is incipient or absent.

Platelet survival times were measured in 11 ambulatory patients within 5 to 21 months of out-of-hospital ventricular fibrillation unassociated with acute myocardial infarction. The mean platelet survival time of 7.7 +/- 2.1 days was shortened when compared with 28 normal controls of similar age (9.0 +/- 1.0 days, p less than 0.03) but not different from 56 other patients with stable angina pectoris (6.85 +/- 1.81 days, p less than 0.2). One patient with a near normal survival time of 7.5 days experienced recurrent ventricular fibrillation without associated acute myocardial infarction 1 month following the platelet survival study. Although one platelet inhibitory agent, sulfinpyrazone, may lower the incidence of sudden cardiac death following acute myocardial infarction, the shortened platelet survival time in the group of patients experiencing out-of-hospital ventricular fibrillation did not distinguish them from others with coronary artery disease.