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Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha- granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta- thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta- thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.

SummaryBlood samples were obtained on four different occasions from 18 cigarette smoking and 34 non-smoking healthy men (age 4Hl9) and analped to assess age- and smoking-associated changes in plasma proteins, btood coagulation and platelet functions. C-ollagen-induced platele t aggregation was signifi cantly inclreased with agng in non-smokers. Significant gfuanges in chronic smokers were increases in platelet count and fibrinogen in plasma; elevation of platelet factor-3 (PF-3) activity in plateletpoor plasma (PPP); increase in serum levels of a1-antitr,?sin, orosomucoid, haptoglobin and properdin factor B; and sfoeftsning of the lag period of collagen-induced platelet aggregation. Filtration of PPP through Mllipore fiIters removed PF-3 membranes. The differen@s in PF-3 activities in fiItered plasma were no longer significant between smokers and non-smokers. Results suggest that chronic smokers have higher levels of acute phase proteins reflecting underlyrng inflammatory processes, and higher levels of PF-3 activity in plasma due to liberation of PF-3 membranes from platelets.

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The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of 14C-5-hydroxytryptamine (14C-5-HT, release I) and P-glucuronidase (release 11) from gel-filtered human platelets. P-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05-0.1 mM). Release of 14C-5-HT, however, was unaffected a t low concentrations of colchicine and only slightly inhibited a t higher concentrations. Inhibition of P-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (l', 5', 20') after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited P-glucuronidase release, but not I4C-5-HT release, induced by trypsin and sodium arachidonate. Binding of 14C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and P-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release I1 and that the cellular mechanisms effecting release I and I1 by platelets differ qualitatively in that the microtubules may facilitate release 11.When blood platelets participate in hemostasis and thrombogenesis, they interact with certain physiological substances and undergo a series of cellular reactions which includes aggregation and a release reaction. In the release reaction the contents of intracellular storage organelles are secreted into the extracellular medium (Holmsen e t al., '69). It has been shown that under certain conditions, the platelet release reaction actually proceeds in two, distinct phases (Holmsen and Day, '70). Release I constitutes the discharge of substances, as adenine nucleotides, calcium and 5-hydroxytryptamine (5-HT), stored in the dense bodies of platelets, and release I1 is marked by the extrusion of lysosomal enzymes and other materials, probably sequestered in platelet a-granules (Day and Holmsen, '71; Holmsen, '75).

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000- dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.