Abstract
Introduction
The use of thromboaspiration in primary percutaneous intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) has offered a unique opportunity to study thrombus composition, its dynamic formation, and architecture in vivo. There has been, however, several limitations, not least the fact that the technique has not yet allowed a precise transversal analysis from one side of the artery to the other, as is done in histological analysis. The dynamic process of intracoronary thrombus formation in STEMI patients is thus still not well understood. Ischemic time was hypothesized to be among the strongest independent correlates of thrombus architecture. In time the platelets are decreasing its proportion and fibrin proportion is increasing (J Silvain, J-P Collet, JW Weisel et al, J Am Coll Cardiol 2011; 57:1359). However, no real report on the internal structures of the in vivo formed thrombi has been shown so far. Therefore, we investigated both the surface and the composition of longitudinally freeze-fractured thrombi.
Methods
Thrombi were collected by PCI from 119 STEMI patients. Out of the patients there were "early comers " (˃12 h from symptom onset; 23 patients) and "late comers" (more than 720 min; 29 patients). The mean age of all patients was 64 years, 70% of patients were males, 51% were smokers, 50% had arterial hypertension, 20% were diabetics and 23% had chronic renal insufficiency.
Scanning electron microscopy; collected thrombi obtained by PCI were thoroughly washed in saline solution and stored in 4% formaldehyde prior dehydration. To reveal the internal structures of the thrombi selected samples were longitudinally freeze fractured in liquid nitrogen and coated with platinum. Samples were examined in SEM Vega Plus TS 5135 (Tescan s.r.o., Brno, Czech Republic). Whole areas of the freeze-fractured thrombi were scanned.
Results and discussion
The thrombus composition of longitudinally freeze-fractured thrombi was compared between groups of "early-comers" and "late-comers. The distribution of the components in the "early comers" thrombi freeze-fracture seemed to be uniform. Platelets were far the main component (about 75 % in proportion) of the "early comers" thrombus, followed by fibrin and other compounds. The amount of red blood cells was negligible (about 2 - 8 %). We did not observe any significant differences between the thrombi in the group of early comers.
Thrombi of the "late-comers" group were composed mainly of red blood cells; platelets and fibrin formed only minority of the thrombi. In contrast to the "early comers" the distribution of the main thrombus components in the "late comers" thrombi was dramatically different between individual parts of the thrombus. The number of platelets and red blood cells varied from 0% to almost 99% and vice versa. It was possible to estimate the initiating place of the thrombus as well as the direction of the growth. Each thrombus could be divided into parts formed mainly either by platelets or by red blood cells. It seems that thrombus develops a regional architecture defined by the extent of platelet activation and packing density. It has been reported that in contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface demonstrating how contracted clots form an impermeable barrier important for hemostasis and wound healing (D Cines, T Lebedeva, J Weisel et al, Blood 2014; 123:1596). Our investigation of the composition of the in vivo formed thrombi supports these results and helps to explain how fibrinolysis is greatly retarded as clots grow and contract.
We have found that on the surfaces of late-comers thrombi fibrin thick fibrils were present. It has been shown that the association of soluble fibrinogen with the fibrin clot results in the reduced adhesiveness of such fibrinogen/fibrin matrices toward leukocytes and platelets (VK Lishko, T Burke, T Ugarova, Blood 2007; 109:1541). Fibrinopeptides A are less accessible for thrombin in surface bound fibrinogen which thus provides additional level of protection of thrombi from premature dissolution (T Riedel, L Medved, JE Dyr, Blood 2011; 117:1700). These findings may have great impact on our knowledge of pathophysiology of the thrombus growth and possible therapeutic consequences related to the time of symptom onset.
Disclosures
No relevant conflicts of interest to declare.
Whole Blood Based Multiparameter Assessment of Thrombus Formation in Standard Microfluidic Devices to Proxy In Vivo Haemostasis and Thrombosis
