A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature

Abstract

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbβ3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.

Figure 1

Measuring plasma protein extravasation in vivo. (A) Representative images of a thrombus formed in a mouse cremaster venule showing platelets (blue) and flash-activated cAlb (green). Each image is taken directly after an activating pulse of 405 nm light. (B) Time course of average platelet (black) and fibrin (white) area (n = 24; ± standard error of the mean [SEM]). (C) For a separate data set we tracked average platelet area (black) and P-selectin–positive area (white) (n = 23; ± SEM). (D) Time course of the combined average of relative cAlb extravasation (n = 47; ± SEM error bars too small to be visualized). (E) A dot plot of the relative cAlb extravasation for each injury at 200 seconds postinjury (n = 47). Plts, platelets; RFU, relative fluorescence units.

Figure 2

Inhibition of α_IIbβ₃ integrin reduces platelet accumulation and vessel sealing. (A) Representative images of thrombi formed in the presence of either vehicle (left) or eptifibatide (right) (20 mg/kg), showing both platelets (blue) and fibrin (red) 3 minutes postinjury. (B) Average platelet area and (C) fibrin area for both the vehicle-treated (black) and eptifibatide-treated (white) thrombi (± SEM). (D) Representative images of cAlb extravasation (green) at both 30 and 180 seconds postinjury for vehicle (top) and eptifibatide treated (bottom) thrombi (platelets are denoted in blue). (E) Time course and (F) dot plot of relative cAlb extravasation for vehicle (black) and eptifibatide (white) treated thrombi (vehicle n = 12, eptifibatide n = 11; ± SEM).

Figure 3

Outside-in signaling drives platelet retraction and vessel sealing. (A) The average platelet area for WT (black) and diYF thrombi (white) (± SEM). (B) Time course (mean ± SEM) and (C) dot plot at 200 seconds postinjury of cAlb extravasation for WT (black) and diYF (white) thrombi (WT n = 24, diYF n = 19).

Figure 4

Thrombin inhibition decreases fibrin accumulation and thrombus size, but not vessel sealing. (A) Representative images of thrombi formed either in the presence of vehicle (left) or thrombin inhibitor hirudin (right) (∼0.7 mg/g) showing platelets (blue) and P-selectin (red) 3 minutes postinjury. (B) Quantification of average platelet area, (C) peak fibrin area, and (D) peak P-selectin area for both vehicle-treated (black) and hirudin-treated (white) thrombi (± SEM). (E) Time course of average cAlb extravasation (± SEM) and (F) dot plot of cAlb extravasation at 200 seconds for both vehicle-treated (black) and hirudin-treated (white) thrombi (vehicle n = 17, hirudin n = 18). P-sel, P-selectin.

Figure 5

ADP drives fully competent core formation and shell recruitment. (A) Quantification of average platelet area, and (B) peak P-selectin–positive area of vehicle (black) and cangrelor-treated (white) thrombi. (C) Average cAlb extravasation time course and (D) dot plot of relative cAlb extravasation at 200 seconds postinjury. (A-D) Vehicle n = 15, cangrelor n = 20; ± SEM). (E) The average platelet area, and (F) peak P-selectin–positive area for vehicle (black) and cangrelor plus hirudin-treated (white) thrombi (± SEM). (G) Time course of relative cAlb extravasation, and (H) dot plot of cAlb extravasation at 200 seconds postinjury. (E-H) Vehicle, black n = 13; cangrelor plus hirudin, white n = 15.

Figure 6

cAlb accumulation in the extravascular space. (A) cAlb accumulation was measured by the relative fluorescence remaining 15 seconds after each activating light pulse for 200 seconds (n = 47, ± SEM). (B) cAlb accumulation was measured for eptifibatide-treated thrombi (white), and vehicle-treated thrombi (black) (vehicle n = 12, eptifibatide n = 11; ± SEM). (C) Representative pseudo-colored images of cAlb intensity 3 minutes postinjury in vehicle-treated (left) and eptifibatide-treated (right) mice. The vessel wall is indicated by the dashed white line and platelets are outlined in black. (D) cAlb accumulation for diYF and WT thrombi, (E) cangrelor-treated, (F) hirudin-treated, and (G) cangrelor/hirudin-treated vs vehicle-treated thrombi (all n values the same as previously reported for the respective data set).

Figure 7

Model of hemostasis in mouse cremaster venules. A model depicting the rate of plasma protein extravasation (L[t]) and dispersion (D[t]) over time, and the resulting impact on the gradient of plasma proteins in the extravascular space.