Platelets are anucleate cells which circulate in the blood in both resting and active forms. Platelets are responsible for the formation of the hemostatic blood that provides the primary defense against hemorrhage following vascular trauma. Upon stimulation and degranulation, activated platelets are recruited into growing thrombi or cleared rapidly from the blood circulation. In the formation of growing thrombi, platelets may contact with and spread upon the subendothelial matrix in a process termed platelet adhesion. The resulting layer of adherent platelets provides the foundation for the hemostatic blood.
There is an increasing understanding of the mechanisms responsible for the activation, secretion, and aggregation of platelets. However, there is still little firm data on the molecular alterations to the platelet membrane that accompany or mediate these three processes. The identification and characterization of proteins that are selectively expressed on the surface of activated platelets or that undergo functional changes during platelet activation may help clarify the steps involved in these processes. However, the changes on the platelet membrane that accompany platelet activation have been partially identified. Stable platelet adhesion requires an interaction between the platelet membrane glycoprotein Ib/IX complex and the multimeric protein von Willebrand factor present in the subendothelial matrix.
Monoclonal antibodies have been used to identify proteins thought to be specifically expressed on the surface of activated platelets (Hsu-Lin et al., J. Biol. Chem. 259:9121-9126 (1984); McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984); Nieuwenhuis et al., Blood 70:838-845 (1987); Gralnick et al., Blook 76 (Suppl. 1):457A (1990); Hayward et al., J. Biol. Chem. 266:7114-7120 (1991)). The best characterized of these proteins, GMP-140 (PADGEM, CD62), is also apparently expressed on the surface of activated endothelial cells (Johnston et al., Cell 56:1033-1044 (1989)). Analysis of the GMP-140 cDNA sequence suggests that the protein is one of the family of lectin-like cell adhesion molecules (Johnston et al., Cell 56:1033-1044 (1989)). GMP-140 also appears to be involved in the interaction between activated platelets and neutrophils or monocytes (Larsen et al., Cell 59:305-312 (1989)). Thus the identification of cellular, activation-dependent antigens in platelets may also help to illuminate the alterations that occur during activation in other cells--e.g. lymphocytes, endothelial cells, neutrophils, and monocytes. Identification of these antigens may also elucidate activation-dependent interactions between different cells or between cells and specific functional ligands.
The identification of other antigens specifically expressed on the surface of activated platelets may help elucidate some of the molecular changes that occur during platelet activation, particularly those responsible for changing the platelet from a quiescent cell to a fully adherent thrombocyte. Perhaps the best example of information derived from the study of platelet surface proteins is the research on the glycoproteins IIb/IIIa. Studies of the GPIIb/IIIa glycoproteins, using specific ligands and monoclonal antibodies, are beginning to elucidate the molecular rearrangements that occur in the GPIIb/IIIa complex, with regard to conformation, density, etc., which are responsible for its conversion to a fully competent "receptor" that mediates platelet aggregation (reviewed in Bennett, J.S., Semin. Hematol. 27:186-204 (1990)). Although these studies have significantly enlarged our grasp of the mechanisms of platelet aggregation, our understanding of other platelet events remains less complete.
The initiating event of many myocardial infarctions (heart attacks) is the hemorrhage into atherosclerotic plaque. Such hemorrhage often results in the formation of a thrombus (or blood clot) in the coronary artery which supplies the infarct zone (i.e., an area of coagulation necrosis which results from an obstruction of blood circulation). This thrombus is composed of a combination of fibrin and blood platelets. The formation of a thrombin-platelet clot has serious clinical ramifications. The degree and duration of the occlusion caused by the fibrin-platelet clot determines the mass of the infarct zone and the extent of damage.
The primary goal of current treatment for myocardial infarction involves the rapid dissolution of the occluding thrombus and the restoration of blood flow (reperfusion). A successful therapy must be capable of sustained effect so that reformation of the clot does not occur after the cessation of therapy. If the fibrin-platelet clot is able to reform, then the affected artery may become reoccluded.
Treatment with thrombolytic agents can often successfully restore coronary blood flow rapidly enough to interrupt myocardial infarction. Unfortunately, the dissolved fibrin-platelet clot has been found to reform after cessation of such thrombolytic therapy in a substantial number of patients. This reformation may result in the reocclusion of the affected blood vessels, and is, therefore, of substantial concern.
A thrombolytic agent is a medicament capable of lysing the fibrin-platelet thrombus, and thereby permitting blood to again flow through the affected blood vessel. Such agents include streptokinase, prourokinase, urokinase, and tissue-type plasminogen activator. See, for example, Ganz et al., J. Amer. Coll. Cardiol. 1:1247-1253 (1983); Rentrop et al. Amer. J. Cardiol. 54:29E-31E (1984); and Gold et al., Amer. J. Cardiol. 53:122C-125C (1984).
Clot lysis is mediated by plasmin vv. Under natural conditions, plasminogen is converted to plasmin by tissue plasminogen activator (t-PA). Activation occurs on the fibrin surface, thus confining proteolytic activity to the appropriate site. After plasmin is set free into the circulation, it is rapidly combined with natural inhibitors. An activation of plasmin is the final and necessary step in the process of protecting against undesirable proteolysis. Such plasmin inhibitors include .alpha.-2 antiplasmin, .alpha.-2 microglobulin and .alpha.-1 antitrypsin, all glycoproteins. .alpha.-2 antiplasmin has a much higher affinity for plasmin than .alpha.-2-macroglobulin and binds specifically to plasmin in a 1:1 ratio. Therefore, clot lysis by the administration of t-PA is limited by the rapid and irreversible inactivation of plasmin by plasmin inhibitors.
All available thrombolytic agents still suffer significant shortcomings, including the need for large doses to be therapeutically efficient, a limited fibrin-specificity, residual toxicity in terms of bleeding complications. Cardiovascular disease is still a major cause of disability. All current agents are associated with thrombolytic reocclusion of blood vessels during or after therapy. Therefore, there remains a need for additional agents which can be utilized alone or in combination with known therapeutic agents. Improvements in thrombolytic therapy which enhance clot lysis, or target the thrombolytic agent to the blood clot are needed.