Figure 5.

Inhibition or absence of platelet (plt) factor XIII-A reduces the extent of clot retraction and enhances the rate of fibrinolysis under flow. (A) Clot retraction was performed in nonsiliconized glass tubes using either normal or factor XIII (FXIII)–deficient (FXIII-def) washed platelets in FXIII-depleted plasma; clotting was initiated for 30 minutes with thrombin (1 U/mL) and CaCl₂ (2 mM), and after 30 minutes, clots were imaged and weighed. Clot weight (g) was measured and compared in normal donors (n = 9) in the presence (gray) and absence (blue) of transglutaminase inhibior (TGI) and FXIII-deficient patients (n = 2 patients; 1 patient was recalled for a second time) in the presence (purple) and absence (green) of TGI. Data represents mean ± SEM of clot weight. Statistical analysis was performed on normal donors and samples with and without TGI compared using paired t-test; ∗∗P < .01. (B) Chandler model thrombi were formed from FXIII-deficient plasma (black dashed line) and the incorporation of either normal healthy washed platelets (blue) in the absence and presence (gray) of TGI or FXIII-deficient washed platelets (green) in the absence and presence (purple) of TGI. Data presented represents the median fluorescence intensity (FU) values normalized to the internal FXIII-deficient plasma control. FXIII-deficient patients (n = 2) and normal healthy controls (n = 2). (C) Thrombus formation under flow was performed in microcapillary chambers and visualized using fluorescence confocal microscopy. Whole reconstituted blood made up from FXIII-depleted plasma, either normal healthy platelets (blue) or FXIII-deficient platelets (green) with the red cells from the same donor in the presence of DiOC6 (4 μg/mL) to label platelets, AF647-labeled fibrinogen (75 μg/mL), and tPA (20 nM). Thrombi were fully formed at a shear rate of 500/second, which was achieved when fibrin and platelets covered the entire surface of the well before flow was switched to a lysis buffer containing 125 nM tPA in Tyrode’s buffer and thrombi were lysed to completion. Data represent the normalized FU values of AF647-fibrin(ogen) from the point at which full coverage was achieved and lysis was initiated to completion, where lysis times are represented in (D). Data represent normal healthy donors (n = 3) and FXIII-deficient patient donors (n = 2; mean ± SEM). (E) Representative images from various time points over the course of fibrinolysis: top images are from samples containing normal platelets in FXIII-depleted plasma and bottom images represent FXIII-deficient platelets in FXIII-depleted plasma. FXIII-deficient patient samples lysed faster, hence the shorter times on representative images. Fibrin(ogen): red; platelets: green. Scale bar represents 100 μm.