Clot Retraction and Its Correlation with the Function of Platelet Integrin αIIbβ3

Abstract

Clot retraction results from retractions of platelet filopodia and fibrin fibers and requires the functional platelet α_IIbβ₃ integrin. This assay is widely used to test the functions of platelets and fibrinogen as well as the efficacy of fibrinolysis. Changes in clot retraction have been found in a variety of hemostatic abnormalities and, more recently, in arterial thrombosis. Despite its broad clinical use and low cost, many aspects of clot retraction are poorly understood. In the present study, we performed two clinical standard clot retraction assays using whole-blood and platelet-rich plasma (PRP) samples to determine how clot retraction correlates with platelet counts and mean volume, the density of α_IIbβ₃ integrin and PLA genotypes, and plasma fibrinogen levels. We found that clot retraction was affected by platelet counts, but not mean platelet volume. It correlated with the surface density of the integrin α_Iibβ₃, but not PLA genotypes. These results indicate that clot retraction measures a unique aspect of platelet function and can serve as an additional means to detect functional changes in platelets.

Keywords: PLA genotypes; clot retraction; integrin αIibβ3.

Figure 1

Correlations between two methods for measuring clot retraction: Whole blood assay (WB) was performed in the absence of anticoagulant and agonist, whereas the PRP assay (PRP) was induced by human α-thrombin using the citrated blood. The percentage of clot retraction was significantly less in the whole blood assay compared to the PRP assay ((A), Mann–Whitney U test, n = 53), and the results from the two methods were closely correlated ((B). Linear regression analysis, n = 53, p < 0.005).

Figure 2

Time course of the PRP clot retraction: Aliquot of 0.6 mL of citrate PRP was incubated with 100 U of human α-thrombin for 15, 30, 60, 90, 120, and 180 min at 37 °C and clot retraction at each time point was measured. The percentage of clot retraction increased proportionally with the incubation time and reached the maximum at 90 min incubation (one-way ANOVA, * p < 0.05 compared to peak level at 180 min, n = 19).

Figure 3

Clot retraction with different anticoagulants: Clot retraction was induced by incubating PRP with 100 U of human α-thrombin for 2 hrs at 37 °C using citrate, PPACK, or heparin as an anticoagulant. The percentage of clot retractions showed no significant differences between the three anticoagulants (one-way ANOVA, n = 14/group).

Figure 4

Correlations between clot retraction and platelet counts: Whole-blood clot retraction was measured and divided into three groups according to their platelet counts: counts of less than 3 × 10⁸/mL, counts between 3–4 × 10⁸/mL, and counts of more than 4 × 10⁸/mL. The percentage of clot retraction was significantly greater in samples whose platelet counts were between 3–4 × 10⁸/mL compared to those with either higher or lower platelet counts (one-way ANOVA, n = 14).

Figure 5

Correlations between clot retraction and plasma levels of fibrinogen: Plasma fibrinogen levels were measured for each donor by an automatic blood analyzer; no overall correlation was found with clot retraction (A, linear regression analysis, n = 33). Samples were further divided into three groups based on their fibrinogen levels: group A had fibrinogen levels less than 250 μg/mL; group B had fibrinogen levels between 250 and 350 μg/mL; group C had fibrinogen levels greater than 350 μg/mL. There was no significant difference in clot retraction among these three groups (B, one-way ANOVA, n = 33, p > 0.05).

Figure 6

Correlations between clot retraction and surface density of the platelet α_IIbβ₃: The numbers of α_IIbβ₃ on platelet surfaces were determined by a flow cytometric method using the monoclonal antibody P2, which recognizes the α_IIbβ₃ complex, but not individual subunits. There was no overall correlation between clot retraction and surface density of the platelet α_IIbβ₃ (A, linear regression analysis, n = 24). The surface density of the α_IIbβ₃ was, however, significantly greater in samples with clot retraction greater than 90% than those with clot retraction of less than 80% (B, Student’s t-test, n = 7–12).