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Recently evidence was provided for a pathway whereby circulating fibrinogen enters megakaryocyte granules by an endocytic mechanism. Synthesis of fibrinogen by megakaryocytes has been reported. To determine the relationship between plasma fibrinogen and alpha-granule fibrinogen in megakaryocytes and platelets, the fibrinogen content of these cells was studied in rats defibrinated by use of Ancrod, a thrombinlike enzyme purified from the venom of Agkistrodon rhodostoma. Unlike thrombin, Ancrod does not induce platelet secretion. Rats were injected with Ancrod (50 units/kilogram body weight) at 8-hour intervals for 5 days. There were no significant changes in platelet counts. Blood from the treated rats failed to clot, and plasma fibrinogen levels were less than 15 mg/dl. Bone marrow from defibrinated rats and untreated control rats was stained immunohistochemically for fibrinogen and two other alpha-granule proteins, albumin and platelet factor 4 (PF4), in plastic-embedded sections. The presence of these three proteins in platelets was detected by Western blots. Only trace amounts of fibrinogen were detected in megakaryocytes and platelets from defibrinated rats, but fibrinogen in control megakaryocytes and platelets was readily demonstrated. However defibrinated and control rats did not differ in albumin and PF4 content in megakaryocytes and platelets. It is concluded that a major portion of rat platelet fibrinogen is derived from plasma by endocytosis by megakaryocytes.

A visual assay to study megakaryocyte platelet release via proplatelet formation in vitro was established. Samples of megakaryocyte-enriched rat bone marrow were incubated (37 C) in RPMI-1640 medium with 15% autologous serum in specially prepared chambers. In the culture system, approximately 6% of megakaryocytes formed proplatelet processes within 24 hours. Inclusion of a heterologous antiplatelet antibody in the culture system inhibited proplatelet formation, compared with that in controls.

Megakaryocytes were isolated from bone marrow from healthy dogs, using a combination of density-gradient centrifugation and polysucrose-velocity sedimentation techniques. The 2-step separation technique resulted in a preparation comprising 30% to 35% megakaryocytes of total nucleated cells. Accessibility to large numbers of viable canine megakaryocytes allowed investigation of platelet release by these cells in short-term cultures. Megakaryocytes were observed to form long cytoplasmic processes that gradually developed segmental constrictions and subsequently fragmented into platelet-sized pieces. Some platelet-sized cytoplasmic pieces of megakaryocytes presumably underwent discoid transformation.

Development of an in vitro visual assay facilitated the study of large numbers of megakaryocytes undergoing proplatelet formation in short-term cultures. Approximately 9% of megakaryocytes formed platelets during a 24-hour period. In the presence of an inhibitor of anaerobic glycolysis (NaF), proplatelet formation was inhibited, whereas inhibitors of respiration (NaCN) did not significantly (P greater than 0.05) decrease proplatelet formation. Presence of the microtubule-disrupting agents colchicine and vincristine sulfate in culture medium inhibited proplatelet formation, whereas the microfilament-disrupting agent cytochalasin B had a less pronounced inhibition.

In a previous study we provide evidence for a circuitous pathway by which circulating plasma proteins enter megakaryocyte granules by an endocytic mechanism and are returned to the circulation in platelets (1987. Proc. Natl. Acad. Sci. USA. 84:861-865). Horseradish peroxidase (40,000 mol wt) was injected into guinea pigs and its uptake into megakaryocyte organelles examined by electron microscopy and cytochemistry. In the present study we tested the ability of guinea pig megakaryocytes to take up intravenously injected albumin, IgG, and fibrinogen. We used two types of proteins to study the endocytic pathway: (a) heterologous human proteins, which were detected immunohistochemically using antibodies that do not crossreact with the native guinea pig counterparts; and (b) human and guinea pig proteins labeled with the small (250 mol wt), inert molecule, biotin, which were detected using an antibody against biotin. We detected all three of the injected proteins in bone marrow megakaryocytes in patterns identical to those of native counterparts. The injected protein consistently appeared in platelets 24 h later and was secreted in response to thrombin. We conclude that there are at least two mechanisms by which guinea pig megakaryocyte granules acquire proteins (a) endogenous synthesis, as demonstrated by others, and (b) endocytosis of plasma proteins synthesized by other types of cells.

To determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for its uptake by electron microscopy and cytochemistry. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare cells. In megakaryocytes, 50% of alpha granules contained HRP between 75 min and 7 hr after injection. At 24 hr, 25% of the megakaryocyte granules were peroxidase-positive, less were positive by 48 hr, and there were none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. Platelet granules also contain HRP by 7 hr after injection, and they can secrete it in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. It is concluded that while some alpha granule proteins are synthesized by megakaryocytes, others may be acquired from plasma by endocytosis. In addition to providing evidence that some of the proteins of alpha granules may be of exogenous origin, this study has allowed the definition of a pathway whereby plasma proteins may be temporarily sequestered in megakaryocytes before reentering the circulation in platelets.

In order to determine whether or not proteins circulating in plasma can be incorporated into megakaryocytes and platelets, horseradish peroxidase (HRP) was injected intravenously into guinea pigs and these cells were examined for uptake by cytochemistry and electron microscopy. Enriched samples of megakaryocytes enabled ultrastructural analysis of large numbers of these rare bone marrow cells. In megakaryocytes, more than 50% of alpha granules contained HRP between 75 minutes and 7 hours after injection. At 24 hours, 25% of the megakaryocyte granules were peroxidase positive; less were so by 48 hours and none at 4 days. Thus, the findings demonstrate that a circulating protein can be endocytosed by megakaryocytes and rapidly packaged into alpha granules. A precipitous drop in circulating platelet numbers was observed 45 minutes after injection. At this time, circulating platelets showed the tracer only on the platelet plasma membrane, and none in platelet granules. Platelet numbers increased to 35% by 7 hours and only the platelet granules contained HRP. These platelets secreted the HRP stored in granules in response to thrombin. Unfortunately, our present studies do not allow us to distinguish between direct endocytosis by the platelet and/or shedding of new platelets from recently labeled megakaryocytes. Our studies are the first to demonstrate an endocytic pathway by which megakaryocytes can incorporate a circulating protein into alpha granules. An important physiologic implication of this endocytic pathway is the possible origin of certain alpha granule proteins from plasma.

A variety of drugs may cause thrombocytopenia. Although it occurs more often than drug-induced anemia it is less well understood because techniques for studying drug-platelet-immune interactions have been unavailable until recently. The mechanisms by which drugs cause thrombocytopenia are varied. Bone marrow suppression or increased peripheral destruction of platelets could be involved. Nonimmunologic as well as immunologic mechanisms may also occur. These different mechanisms of drug-induced thrombocytopenia are reviewed. Diagnostic methods and treatment are also summarized.

A technique to isolate megakaryocyte proplatelet processes from blood of rats' hearts, using colloidal silica coated with polyvinylpyrrolidone density gradient, was developed. The proplatelet concentration in blood from right ventricles was significantly higher (P less than 0.001) than that in blood from left ventricles in healthy rats, as well as in rats with induced acute blood loss. The proplatelet concentration of blood from the heart, 24 hours after acute blood loss was induced was significantly (P less than 0.001) increased, indicating that platelet production was accelerated. The demonstration of proplatelets entering the pulmonary circulation indicates platelet release via proplatelet formation. Seemingly, proplatelets are fragmented in the lungs at predesignated locations along the proplatelet process.