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We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At 2 25 gg/ml, PF4 inhibited megakaryocyte colony formation -80% in unstimulated cultures, and -58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 gg/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue fithromboglobulin (,f-TG) peptide and native fl-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1±3.0 vs. 4.2±1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6±1.5 vs. 3.9±2.3; P < 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc, and c-myb), and non-growth-regulated (fi2-microglobulin) genes. At high concentrations of native PF4 (50 ,gg/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb.The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or ,B-TG peptides for 18 h and effect on Factor V steadystate mRNA levels was determined in 600 individual cells by in situ hybridization. fl-TG peptide had no effect on FV mRNA levels, whereas a -60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to 2 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may funcAddress reprint requests to Dr.

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein lIb/llla complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 /lM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IlIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of j-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of a granules and ,3-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that distintegrins may be potential candidates for platelet protection during cardiopulmonary bypass. (Circulation 1990;82:261-273) Cardiopulmonary bypass involves extensive contact between blood and synthetic surfaces of the membrane oxygenator. Bypass produces thrombocytopenia,1"2 platelet fragmen-

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