Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t-PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet-induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.     

Platelet secretion during clot retraction

The precise route serving as the pathway for extrusion of products contained in platelet storage organelles following cell activation has been controversial for many years. Some workers believe that the granules and dense bodies fuse together to form secretion vacuoles that move to the exposed cell surface, fuse with it and extrude contained products to the outside. Others suggest that the organelles fuse with channels of the open canalicular system (OCS) and deposit their contents which are subsequently secreted to the exterior. The controversy has been difficult to resolve because nearly all workers have used platelets in suspension to arrive at diverse conclusions. The present study has used ethylenediamine tetracetic acid (EDTA)-treated platelets and isometric clot retraction as a model system, and either polylysine or tannic acid to define the pathway used for secretion. Results indicate that secretion takes place in EDTA platelets under isometric tension by fusion of granules to OCS channels and passage of products through these conduits to the cell exterior. There is no evidence to suggest the organelles form secretion vacuoles or extrude their products by fusion with the inside of the exposed surface membrane.     

Platelet secretion during clot retraction

The precise route serving as the pathway for extrusion of products contained in platelet storage organelles following cell activation has been controversial for many years. Some workers believe that the granules and dense bodies fuse together to form secretion vacuoles that move to the exposed cell surface, fuse with it and extrude contained products to the outside. Others suggest that the organelles fuse with channels of the open canalicular system (OCS) and deposit their contents which are subsequently secreted to the exterior. The controversy has been difficult to resolve because nearly all workers have used platelets in suspension to arrive at diverse conclusions. The present study has used ethylenediamine tetracetic acid (EDTA)-treated platelets and isometric clot retraction as a model system, and either polylysine or tannic acid to define the pathway used for secretion. Results indicate that secretion takes place in EDTA platelets under isometric tension by fusion of granules to OCS channels and passage of products through these conduits to the cell exterior. There is no evidence to suggest the organelles form secretion vacuoles or extrude their products by fusion with the inside of the exposed surface membrane.     

Control of clot lysis by gene transfer

Intravascular clot formation is a local process that can result in serious clinical consequences, including limb loss and death. Gene transfer and expression of recombinant plasminogen activators in the endothelial cells of the vessel wall offer an attractive approach to the enhancement of local fibrinolytic activity. In vitro studies have demonstrated that endothelial cell fibrinolytic activity can be increased by gene transfer of either secreted or cell surface-anchored plasminogen activators. Future work will define the ability of gene transfer to facilitate clot lysis in vivo.     

Reptilase clot retraction induced by electrical stimulation.

Retraction of platelet rich plasma clotted by reptilase is induced by electrical stimulation. Optimal retraction is obtained by stimuli, applied for more than 4 min, with the following characteristics: intensity = 150 volts, duration = 50 msec each, frequency = 10/sec. Electrically induced reptilase clot retraction is shown to be inhibited by EDTA, EGTA, methyl-xanthines, PGE1, acetylsalicylic acid, indomethacin, but not by apyrase or by phosphoenolpyruvate-pyruvate kinase and MgCl2. The results indicate that electrical stimulation induces retraction of PRP clotted by reptilase by triggering off an increased availability of Ca2+ in the intracellular space.     

Fibrin clot formation and lysis: basic mechanisms

The hemostatic balance, introduced more than 40 years ago, addresses the components and reactions involved in fibrin turnover. Fibrin is placed in the core of this delicate balance. Defects in the mechanisms responsible for fibrin turnover might lead to thrombosis or bleeding, and fibrin consequently is an important substrate in the physiology of hemostasis. This review describes the components and processes involved in fibrin formation and fibrin degradation. Particular emphasis is put on the reactions involved in the conversion of fibrinogen to fibrin, the polymerization of fibrin molecules induced by coagulation factor XIII (FXIII), and the degradation of fibrinogen and fibrin mediated by plasmin and elastase. Furthermore, factors influencing fibrin structure and fibrin breakdown are addressed; in particular polymorphisms in the genes coding for fibrinogen and FXIII, but also the physical and biochemical conditions in which fibrin is formed. The past decades have produced a bulk of biochemical publications reviewing fibrin turnover and fibrin structure, and it has been shown that alterations in fibrin structure are important for the development of various disease conditions, whereas, the architecture of fibrin can be modified by certain drugs and chemical compounds. However, these topics deserve increased attention in clinical settings. Of particular importance might be more detailed clinical studies that review the influence of polymorphisms in the genes coding for the key factors involved in fibrin metabolism on the development of hemostatic diseases, but also the role of elastase-induced fibrin degradation deserves increased attention.