ADP Receptor Antagonists and Converting Enzyme Systems Reduce Platelet Deposition onto Collagen

 

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Summary

The effect of the ADP receptor antagonists ATP and adenosine 5’-(β, γ-methylene)triphosphate (AMP-PCP), and the ADP-utilizing enzyme systems creatine phosphokinase/creatine phosphate (CPK/CP) and pyruvate kinase/phosphoenol pyruvate (PK/PEP) on platelet deposition onto type I collagen was examined. An in vitro perfusion system was used, which allowed continuous visualization of the deposition of fluorescently labelled platelets. This system also provided well-controlled rheology, precise quantification of deposition, and allowed the use of heparinized whole human blood (3 u/ml). Heparinization at this level permits the local generation of thrombin near surface platelet aggregates. The contribution of ADP is thus studied with the combined effects of thrombin, thromboxane A₂, and other aggregating agents present. Results from these studies indicate that ATP was capable of inhibiting deposition by 60% at 1 pM and 90% at 5 μM (whole blood cone.). AMP-PCP inhibited deposition in a dose dependent manner with a K_i of approximately 80 μM and a maximum inhibition of 60%. Inhibition by CPK/CP was measured at 20, 40, and 60 u/ml, with approximately 45% inhibition achieved for the latter two concentrations. PK/ PEP at 60 u/ml resulted in 70% inhibition. These results support a role for ADP in mediating platelet recruitment in thrombus growth on collagen. Previous work utilizing animal bleeding times supports this conclusion; the present study demonstrates that this role is not dependent upon endothelial or vasoconstrictive effects. Intraplatelet cAMP levels were raised with respect to controls upon exposure to ATP at 8.3 μM (p <0.025), and 15 μM (p <0.005), as well as AMP-PCP at 42-500 μM (p <0.005). Since increasing intraplatelet cAMP may affect platelet function, these results raise another possible interpretation for some of our platelet deposition data as well as that of previous experimenters. However, since ATP at 1 μM was not shown to affect intraplatelet cAMP, it is believed that at least part of the ATP-mediated inhibition was due to ADP receptor competition.

• References

• 1 Haslam RJ. Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids. Nature 1964; 202: 765-768
  CrossrefPubMedGoogle Scholar
• 2 Haslam RJ. Mechanisms of blood platelet aggregation. In: Physiology of Hemostasis and Thrombosis. Johnson SA, Seegers WH. (eds) Charles C Thomas; Springfield, IL: 1967. pp 88-112
  Google Scholar
• 3 Mills DCB, Robb IA, Roberts GCK. The release of nucleotides, 5-hydroxytryptamine and enzymes from human blood platelets during aggregation. J Physiol (London) 1968; 195: 715-729
  CrossrefPubMedGoogle Scholar
• 4 Weiss HJ. Platelet physiology and abnormalities of platelet function (First of two parts). N Engl J Med 1975; 293: 531-541
  CrossrefPubMedGoogle Scholar
• 5 Marcus AJ. The role of lipids in platelet function: with particular reference to the arachidonic acid pathway. J Lipid Res 1978; 19: 793-826
  PubMedGoogle Scholar
• 6 Zucker MB. Platelet function. In: Hematology. Williams WJ, Beutler E, Erslev AJ, Lichtmann MA. (eds) McGraw-Hill; New York: 1983. pp 1176-1134
  Google Scholar
• 7 Emms H, Lewis GP. The roles of prostaglandin endoperoxides, thromboxane A2, and adenosine diphosphate in collagen-induced aggregation in man and the rat. Br J Pharmacol 1986; 87: 109-115
  CrossrefPubMedGoogle Scholar
• 8 Hubbell JA, Mclntire LV. Platelet active concentration profiles near growing thrombi: a mathematical consideration. Biophys J 1986; 50: 937-945
  CrossrefPubMedGoogle Scholar
• 9 Folie BJ, Mclntire LV. Mathematical analysis of mural throm-bogenesis: concentration profiles of platelet-activating agents and effects of viscous shear flow. Biophys J 1989; 56: 1121-1141
  CrossrefPubMedGoogle Scholar
• 10 Hubbell JA, Mclntire LV. Visualization and analysis of mural thrombogenesis on collagen, polyurethane, and nylon. Biomaterials 1986; 7: 354-363
  CrossrefPubMedGoogle Scholar
• 11 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-929
  PubMedGoogle Scholar
• 12 Mustard JF, Packham MA. Factors influencing platelet function: adhesion, release and aggregation. Pharmacol Rev 1970; 22: 97-187
  PubMedGoogle Scholar
• 13 Macfarlane DE, Mills DCB. The effects of ATP on platelets: evidence against the central role of released ADP in primary aggregation. Blood 1975; 46: 309-320
  PubMedGoogle Scholar
• 14 Grabowski EF, Franta JT, Didisheim P. Platelet aggregation in flowing blood in vitro. II. Dependence of aggregate growth rate on ADP concentration and shear rate. Microvasc Res 1978; 16: 183-195
  CrossrefPubMedGoogle Scholar
• 15 Zawilska KM, Born GVR, Begent NA. Effect of ADP-utilizing enzymes on the arterial bleeding time in rats and rabbits. Br J Haematol 1982; 50: 317-325
  CrossrefPubMedGoogle Scholar
• 16 Born GVR, Cusack NJ, Darwin C, McClure M. Prolongation of bleeding time in rats by ADP receptor antagonists. J Physiol (London) 1986; 381: 22P
  PubMedGoogle Scholar
• 17 McClure MO, Kakkar A, Cusack NJ, Born GVR. Evidence for the dependence of arterial haemostasis on ADP. Proc R Soc London 1988; B234: 255-262
  CrossrefPubMedGoogle Scholar
• 18 Haslam RJ, Cusack NJ. Blood platelet receptors for ADP and for adenosine. In: Purinergic Receptors: Receptors and Recognition, Series B. Burnstock G. (ed). Vol 12 Chapman and Hall; London: 1981. pp 221-285
  Google Scholar
• 19 Cusack NJ, Hourani SMO. Adenosine 5’-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors. Br J Pharmacol 1982; 76: 221-227
  CrossrefPubMedGoogle Scholar
• 20 Cusack NJ, Hourani SMO, Welford LA. Characterisation of ADP receptors. In: Mechanisms of Stimulus-Response Coupling in Platelets. Westwick J, Scully MF, MacIntyre DE, Kakkar VV. (eds) Plenum Press; New York: 1985. pp 29-39
  Google Scholar
• 21 Tschopp TB, Baumgartner HR. Enzymatic removal of ADP from plasma: unaltered platelet adhesion but reduced aggregation on subendothelium and collagen fibrils. Thromb Haemostas 1976; 35: 334-341
  PubMedGoogle Scholar
• 22 Soslau G, Parker J. Modulation of platelet function by extracellular adenosine triphosphate. Blood 1989; 74: 984-993
  PubMedGoogle Scholar
• 23 Sage SO, Reast R, Rink TJ. ADP evokes biphasic Ca²⁺ influx in fura-2-loaded human platelets. Biochem J 1990; 265: 675-680
  CrossrefPubMedGoogle Scholar
• 24 Lerea KM, Glomset JA, Krebs EG. Agents that elevate cAMP levels in platelets decrease thrombin binding. J Biol Chem 1987; 262: 282-288
  PubMedGoogle Scholar
• 25 Puri PN, Zhou F, Colman RW. Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors. Biochem Biophys Res Commun 1989; 162: 1017-1024
  CrossrefPubMedGoogle Scholar
• 26 Burke JM, Ross R. Synthesis of connective tissue macromolecules by smooth muscle. Int Rev Connect Tissue Res 1979; 8: 119-157
  PubMedGoogle Scholar
• 27 McCullagh KA, Balian G. Collagen characterisation and cell transformation in human atherosclerosis. Nature 1975; 258: 73-75
  CrossrefPubMedGoogle Scholar
• 28 Wagner WR, Hubbell JA. Local thrombin synthesis and fibrin formation in an in vitro thrombosis model result in platelet recruitment and thrombus stabilization on collagen in heparinized blood. J Lab Clin Med 1990; 116: 636-650
  PubMedGoogle Scholar
• 29 Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci USA 1988; 85: 3184-3188
  CrossrefPubMedGoogle Scholar
• 30 Jang IK, Gold HK, Ziskind AA, Leinbach RC, Fallon JT, Collen D. Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition. Circulation 1990; 81: 219-225
  CrossrefPubMedGoogle Scholar
• 31 Hauptmann J, Kaiser B, Nowak G, Sturzebecher J, Markwardt F. Comparison of the anticoagulant and antithrombotic effects of synthetic thrombin and factor Xa inhibitors. Thromb Haemostas 1990; 63: 220-223
  PubMedGoogle Scholar
• 32 Desai NP, Hubbell JA. The short-term blood biocompatibility of poly(hydroxyethyl methacrylate-co-methyl methacrylate) in an in vitro flow system measured by digital videomicroscopy. J Biomater Sci Polymer Edn 1989; 1: 123-146
  CrossrefPubMedGoogle Scholar
• 33 Hubbell JA, Mclntire LV. Technique for visualization and analysis of mural thrombogenesis. Rev Sci Instrum 1986; 57: 892-897
  CrossrefPubMedGoogle Scholar
• 34 Cazenave JP, Packham MA, Mustard JF. Adherence of platelets to a collagen-coated surface: development of a quantitative method. J Lab Clin Med 1973; 82: 978-990
  PubMedGoogle Scholar
• 35 Adams GA, Brown SJ, Mclntire LV, Eskin SG, Martin RR. Kinetics of platelet adhesion and thrombus growth. Blod 1983; 62: 69-74
  PubMedGoogle Scholar
• 36 Sakariassen KS, Baumgartner HR. Axial dependence of platelet-collagen interactions in flowing blood. Arteriosclerosis 1989; 9: 33-42
  CrossrefPubMedGoogle Scholar
• 37 Coade SB, Pearson JD. Metabolism of adenine nucleotides in human blood. Circ Res 1989; 65: 531-537
  CrossrefPubMedGoogle Scholar
• 38 Huang EM, Detwiler TC. Reassessment of the evidence for the role of secreted ADP in biphasic platelet aggregation. J Lab Clin Med 1980; 95: 59-68
  PubMedGoogle Scholar
• 39 Nunn B, Chamberlain PD. Further evidence against the validity of using an ADP-removing enzyme system (CP/CPK) for demonstrating the role of secreted ADP in platelet activation. Thromb Res 1983; 30: 19-26
  CrossrefPubMedGoogle Scholar
• 40 Gaarder A, Jonsen J, Laland S, Hellem A, Owren PA. Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets. Nature 1961; 192: 531-532
  CrossrefPubMedGoogle Scholar
• 41 Bom GVR, Kratzer MAA. Source and concentration of extracellular adenosine triphosphate during haemostasis in rats, rabbits and man. J Physiol (London) 1984; 354: 419-429
  CrossrefPubMedGoogle Scholar
• 42 Kratzer MAA, Born GVR. Free ATP in blood during haemorrhage. Thromb Haemostas 1981; 46: 393
  PubMedGoogle Scholar
• 43 Bell DN, Spain S, Goldsmith HL. The effect of red blood cells on the ADP-induced aggregation of human platelets in flow through tubes. Thromb Haemostas 1990; 63: 112-121
  PubMedGoogle Scholar
• 44 Bom GVR, Wehmeier A. Inhibition of platelet thrombus formation by chlorpromazine acting to diminish haemolysis. Nature 1979; 282: 212-213
  CrossrefPubMedGoogle Scholar
• 45 Alkhamis TM, Beissinger RL, Chediak JP. Artificial surface effect on red blood cells and platelets in laminar shear flow. Blood 1990; 75: 1568-1575
  PubMedGoogle Scholar
• 46 Adams GA, Feuerstein IA. Platelet adhesion and release: interfacial concentration of released materials. Am J Physiol 1981; 240: H99-H108
  PubMedGoogle Scholar
• 47 Wagner WR, Hubbell JA. Evidence for a role in thrombus stabilization for thromboxane A₂ in human platelet deposition on collagen. J Lab Clin Med. (in press)
  PubMedGoogle Scholar