From Fibrinogen to Fibrin: Dynamic Light Scattering to Probe the Mechanism of Protofibril Assembly Into Fibers.

Abstract

AbstractAbstract 1156The conversion from fibrinogen to fibrin, and fibrin clot structure and function, are important problems in cardiovascular research. The mechanism of fibrin clot formation remains undefined after more than 50 years of study. We have developed a new method to monitor polymerization and examine the products formed prior to gelation. We monitored polymerization by dynamic light scattering (DLS), a sensitive method to measure formation of small complexes. We “stopped” thrombin-catalyzed polymerization at different phases by crosslinking the polymers with formaldehyde and analyzed the products by DLS, agarose gel electophoresis (AGE) and transmission electron microscopy (TEM). DLS of the formaldehyde fixed products showed the products were stable and were analogous to the polymerization reactions with about a 2 minute delay in the average radius. As shown in the table, AGE showed a decrease in the numbers of small molecules and the appearance of larger molecules with polymerization. Preliminary TEM results showed a similar progression with AGE and clearly displayed the polymerization process from fibrin monomers, oligomers, protofibrils, then fibers. About 6% protofibrils were observed at 2 minutes and continuously increased with polymerization. Small amounts of fibers appeared from 4 minutes, and increased both in sizes and numbers with polymerization. At 10 minutes, about 8% fibers were observed and the length of large fibers reached around 3 μm. TEM also showed that after 7 minutes, the polymerization products were relatively heterogeneous with a high dispersity of sizes. With these experiments we followed the polymerization process step by step and determined the changes between steps by comparing adjacent-time products. Additionally, our laboratory specializes in synthesizing recombinant variant fibrinogens that have proven useful in studying functional attributes of fibrinogen. By DLS, we examined polymerization of variants in calcium binding sites: the single substitution gE132A (in the b2 calcium site) and the double substitutions gD318A,D320A (in the high affinity calcium site) and gD298A,D301A (in the g2 calcium site). For gD298A,D301A the DLS profile was similar to normal fibrinogen with a slower formation of protofibrils, as expected for the slightly prolonged lag time. For gE132A DLS showed a more rapid rise and higher plateau than normal fibrinogen. The initial polymerization is the same as normal fibrinogen shown in DLS, indicating the rate of protofibril formation for gE132A is the same as normal. After protofibril formation, the rapid rise in DLS for gE132A preceded the rapid rise for normal fibrin, indicating that assembly of the variant protofibrils into fibers was faster than normal. For gD318A,D230A fibrinogen we saw a slight change in DLS, such that the average radius increased 2-fold in 8.5 hours. This change in radius was not different after 13 hours, indicating a stable complex was formed. Further experiments are needed to characterize this complex. Considered together, these initial studies show that DLS can provide additional insight into the influence of structure on polymerization. The comparison of the normal fibrinogen to variant fibrinogens will help us to better understand the role of functional sites in polymerization and identify the residues of fibrinogen that influence protofibril formation and lateral aggregation.Table 1AGE result of polymerization products crosslinked by formaldehyede at different timepoints.ConstituentPercent composition10 min0 min2 min4 min7 minmonomer97.4%84.4%83.7%80.3%56.8%dimer2.6%14.3%12.5%11.7%22.1%trimer1.3%3.8%3.9%13.0%tetramer2.5%3.6%pentamer1.6%3.5%hexamer1.0% Disclosures:No relevant conflicts of interest to declare.