Effects of Plasmin on von Willebrand Factor and Platelets: A Narrative Review

Plasmin is the major fibrinolytic protease responsible for dissolving thrombi by cleavage of its primary substrate fibrin. In addition, emerging evidence points to other roles of plasmin: (1) as a back-up for ADAMTS13 in proteolysis of ultra-large von Willebrand factor (VWF) multimers and (2) as an activator of platelets. Although the molecular mechanisms of fibrinolysis are well defined, insights on the effects of plasmin on VWF and platelets are relatively scarce and sometimes conflicting. Hence, this review provides an overview of the literature on the effects of plasmin on VWF multimeric structures, on VWF binding to platelets, and on platelet activation. This information is placed in the context of possible applications of thrombolytic therapy for the condition thrombotic thrombocytopenic purpura.


Introduction
Plasmin is the key protease of the fibrinolytic system. During clot development, fibrin deposition elicits generation of plasmin from plasminogen, resulting in the dissolution of the fibrin clot. 1 Although many studies have focused on mechanisms of plasmin-mediated fibrin cleavage (reviewed by, among others, Cesarman-Maus and Hajjar 2 ), there is a relative gap in knowledge on the effects of plasmin on other components that form the primary blood clot/thrombus. Recently, there has been increasing attention for a potential role of plasmin in the treatment of disorders associated with spontaneous von Willebrand factor (VWF)-platelet aggregation, such as thrombotic thrombocytopenic purpura (TTP). 3,4 This review provides an overview of the literature on the effects of plasmin on VWF and platelets. After a brief introduction into the structure and functions of plasmin, the evidence supporting a role for plasmin in degradation of VWF multimers and VWF-platelet complexes is presented. This is followed by a summary of existing literature on the effect of plasmin on platelet activation through the platelet GP1b, GPIIb/IIIa, and thrombin receptors.
terminal heavy chain (12-65 kDa) and a C-terminal light chain (25 kDa). 8 Activation of plasminogen is inhibited by plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). 9,10 The primary substrate of plasmin is fibrin, which regulates its own degradation by binding both plasminogen 11 and tPA 12 on its surface, thereby localizing and enhancing plasmin generation. This binding of plasminogen to fibrin can be blocked by thrombin-activatable fibrinolysis inhibitor (TAFI). 13 Importantly, plasmin can also cleave both tPA and uPA, transforming them from single chain to more active two-chain polypeptides, thereby forming a positive-feedback loop (►Fig. 2). 2 Plasmin is rapidly inhibited by its major inhibitor α2-antiplasmin (α2-AP), and to a lesser extent by α2-macroglobulin (α2M), unless it remains bound to fibrin or to its cell surface receptors (►Fig. 2). 14 Plasmin does not only cleave fibrin, it also possesses exceptionally broad specificity for target substrates, targeting among others coagulation factors V, 15 VIII, 16 IX, 17 X, 18 TFPI, 19 and fibrinogen; 20 as well as the VWF cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) 21 (►Table 1).

Structure and Regulation of von Willebrand Factor
VWF is a large plasma glycoprotein with essential functions in hemostasis. VWF circulates in plasma, at a concentration of approximately 10 μg/mL, 22 in the form of multimers that comprise a varying number of VWF monomers (250-270 kDa). 23 Thus, the molecular weight of VWF multimers ranges from 500 to 20,000 kD, depending on the number of subunits. 24 Each VWF monomer possesses several domains, namely, the D'D3, A1, A2, A3, D4, C1 to C6, and CK domains. 25 Under normal blood flow conditions, VWF adopts a globular conformation 26 that can bind coagulation FVIII, prolonging its lifetime in the circulation. 27,28 Vascular injury induces activation of endothelial cells 29 and platelets, 30 resulting in the release of hyperreactive ultra-large (UL) VWF multimers from Weibel-Palade bodies or α-granules, respectively (►Fig. 3A). These UL-VWF multimers are subjected to rapid proteolysis by ADAMTS13. The resulting ADAMTS13-cleaved VWF multimers are less thrombotic, but can still support normal hemostasis. 31 By inducing vasoconstriction, vascular injury also increases the hydrodynamic force, resulting in elongation of VWF multimers. 32 Unfolded VWF can bind to subendothelial collagen (via A1 and A3 domains) 26 and interact with platelets through GPIbα 33 (via VWF A1 domain 34 ) which slows down the platelets through GPIIb/IIIa (via VWF C4 domain 35 ) resulting in platelet activation, shape change, expression of activated integrins, and secretion of autocrine agents present on platelets.

Plasmin: Candidate Protease for von Willebrand Factor
Rationale for Plasmin as Alternative for ADAMTS13 Deficiency in ADAMTS13 results in TTP, a disorder of thrombotic microangiopathy, characterized by an abnormal persistence of UL-VWF multimers. 36 Similar to the pathology in systemic inflammation/sepsis, thrombotic microangiopathies such as TTP are associated with acute dysfunctional endothelial cell activation, indicated by up to fourfold increased VWF antigen (VWFAg) and VWF propeptide (VWFpp). 37 Interestingly, TTP episodes occur sporadically in patients with hereditary ADAMTS13 deficiency, 38 while patients with acquired (autoantibody-mediated) ADAMTS13 deficiency can achieve clinical remission despite being severely depleted of ADAMTS13. 39 Hence, ADAMTS13 levels do not correlate with disease severity during the acute phase, suggesting the existence of regulatory mechanisms/factors other than ADAMTS13 that modulate the presentation of thrombotic microangiopathy. 39,40 However, during clinical remission, persistent ADAMTS13 deficiency is an established risk factor for clinical relapse. 41,42 Plasmin is a candidate enzyme that may serve as an alternative, or back-up, for ADAMTS13 to cleave VWF multi-mers. 4 The concentration of plasmin in the thrombus is above 10 nM and the environment of the thrombus protects it, at least partially, from inactivation by plasma inhibitors, most importantly α2-AP. 43 Early studies report cleavage by plasmin of the multimeric structure of VWF to lower molecular weight fragments, 44 mainly within disulfide loops, 45 resulting in loss of factor VIII 46 and VWF (ristocetin cofactor) activity. 47 An overview of reported effects of plasmin on VWF and the respective experimental concentrations used is given in ►Table 2.

Mechanisms of VWF Proteolysis by Plasmin
Plasmin, locally produced during fibrinolysis, can release VWF within minutes from native subendothelium. Thereby, proteolytic fragments are generated with potential activity in terms of platelet aggregation. 48 VWF is a relatively poor plasmin substrate compared with fibrinogen. In fact, VWF protects fibrinogen (but not fibrin) against degradation by Fig. 2 Overview of fibrinolysis. Single-chain (sc) tPA and uPA are secreted from endothelial cells and from renal epithelium, monocytes/ macrophages, or endothelial cells, respectively. Both tPA and uPA can be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). Once plasmin is generated, it converts single-chain tPA and uPA to two-chain (tc) forms. Plasminogen is converted into the active protease plasmin primarily by tc-tPA (with fibrin as a cofactor) or tc-uPA. Plasmin cleaves fibrin to fibrin degradation products (FDPs), which can be inhibited by TAFI. Plasmin itself is inhibited by α2-antiplasmin (α2-AP) and α2-macroglobulin (α2-M). Effects of Plasmin on von Willebrand Factor and Platelets van der Vorm et al. e220 plasmin, thereby preserving procoagulant activity in plasma and its adhesive role in platelet thrombi. 49 This might indicate that the presence of VWF in platelet-rich thrombi may contribute to their resistance to thrombolytic therapy. 49 Plasmin has limited affinity for binding to and cleavage of globular VWF, but shear-or denaturant-(e.g., ristocetin) induced unfolding and consequent exposure of the VWF A1 domain strongly enhance this process. 3,4,50 Of note, plasminbinding sites and plasmin proteolysis sites within VWF are distinct. Under shear stress, conformational changes in A1A2 facilitate plasmin binding to lysine-rich regions 1405 KKKK 1408 in the VWF A1 domain, which subsequently enables specific plasmin cleavage at the K1491-R1492 peptide bond within the VWF A1-A2 linker region. 3,4 VWF glycans play an important role in modulating VWF cleavage by plasmin. 3,51 First, desialylation of VWF enhances proteolysis by plasmin while it inhibits ADAMTS13 proteolysis. 3,52,53 Second, susceptibility of VWF to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3. More precisely, N-linked glycans expressed at N1515 and N1574 within the A2 domain protect VWF against proteolysis by both plasmin and ADAMTS13. 3,54,55 Finally, ABO(H) blood group determinants modulate VWF susceptibility to proteolysis by ADAMTS13 (O ! B > A ! AB) but do not influence plasmin-mediated cleavage. 3,56

Disaggregation of VWF-platelet Complexes in the Presence of Plasmin
In the complete absence of ADAMTS13, as in TTP, platelets as well as UL-VWF are present in the circulation (►Fig. 3B). However, obstructive microthrombi do not form spontaneously until a triggering event, for instance an infection, occurs. At that moment, vascular endothelial cells are the first to react on differences in oxygenation and respond to hypoxia through expression of extracellular uPA receptor. 57 Subsequently, uPA-dependent plasmin formation on endothelial cells results in degradation of platelet-VWF complexes. 4 Furthermore, exogenously added thrombolytic agents degrade platelet-VWF complexes with equal efficacy and can be securely controlled by the administration of lysine analogues. 4 This may have important clinical implications, as thrombolytic agents may thus have therapeutic value in the treatment of TTP. 4 In fact, Tersteeg et al found that a single dose (10 U/mL, corresponding to $20% of the loading dose given for pulmonary embolism) of the plasminogen (human, injected at 20 mg/ kg) activator streptokinase was sufficient to attenuate symptoms of TTP and correct the thrombocytopenia in an ADAMTS13-deficient mouse model. Importantly, no evidence of bleeding or a perturbed secondary hemostasis was observed after this treatment. 4 Hence, further studies on the safety and efficacy of therapeutic application of thrombolytic agents for TTP are warranted.

Plasmin and Platelets
A comprehensive overview of platelet adhesion, activation, and aggregation was reviewed by Broos et al. 58 Key findings on the effects of plasmin on platelets reported in literature and the experimental concentrations of plasmin used are summarized in ►Table 3.

(In)activation of Platelets by Plasmin
Under physiological conditions, plasmin activity is restricted to the proximity of the thrombus by plasma proteinase inhibitors. In stroke or deep vein thrombus formation, treatment with thrombolytic drugs can lead to free active plasmin in the circulation. 59,60 Sometimes, after a successful reperfusion, reocclusion of the damaged vessel occurs and marked platelet activation can be detected. 20,61-66 Glu-plasminogen Lys62 Arg68 Lys77 Wiman 102 Wiman and Wallén 103 Violand and Castellino 104 Factor V Arg348 Lys1656 Arg1765 Lys1827 Omar and Mann 15 Lee and Mann 105  Effects of Plasmin on von Willebrand Factor and Platelets van der Vorm et al. e221 In the early 1970s, Niewiarowski et al were the first to report reversible plasmin-induced platelet aggregation, accompanied by a significant granule release. 67,68 In contrast, plasmin treatment could also reduce platelet aggregation after stimulation with thrombin, collagen, or adenosine diphosphate (ADP). 67,69 These results already suggested a dual effect of plasmin depending on plasmin concentration, temperature, exposure time, and platelet environment.
Plasmin, at concentrations !1 caseinolytic unit [CU]/mL causes platelet degranulation and aggregation in in vitro studies using isolated, plasma-free platelets. 61,[70][71][72][73][74] To compare, the amount of plasminogen in 1 mL of normal plasma corresponds to 3 to 4 CU, 75 which amounts to a concentration of 20.3 AE 2.6 mg/100 mL or 80 µM of plasmin. The clinical dose of r-tPA used for treatment of ischemic stroke (0.9 mg/ kg for 1 hour) resulted in the generation of 1.6 CU/mL  (2) Alternatively, a proportion of UL-VWF may remain transiently bound to the endothelial surface during exocytosis and unfold to a stretched conformation in response to shear. This conformation exposes the VWF A2 domain, enabling ADAMTS13 to cleave and release VWF into the circulation, where it adopts a globular conformation. (3) During passage through the microvasculature, globular UL-VWF in free circulation may unravel partially, allowing for cleavage of the largest, most thrombogenic multimers to smaller multimers that do not spontaneously interact with platelets. (4) At sites of vessel damage, plasma VWF will bind to exposed subendothelial collagen and subsequently, VWF unravels, and recruits platelets. The presence of collagen and thrombin induces rapid platelet activation, resulting in platelet plug formation. (B) Plasmin as an alternative protease for ADAMTS-13 to cleave VWF multimers. (1) Low or reduced ADAMTS13 levels/activities result in the loss of plasma VWF processing. Under these circumstances, platelets become bound to transiently unraveled VWF, (2) leading to accumulation of VWF-platelet aggregates that occlude the microvasculature, as seen in patients presenting with TTP.   Plasmin concentrations that did not affect platelet shape change, release, or aggregation (< 1.0 CU/mL) caused a doseand time-dependent inhibition of platelet aggregation in response to thrombin, ionophore A23187, and collagen. Complete loss of aggregation occurred at 0.1-0.5 CU/mL of plasmin. Plasmin produces a small rise in platelet cytosolic Ca 2þ and a tyrosine kinase-dependent enhancement of Ca 2þ turnover.  88 Desensitization of PAR1 has no effect on plasmin-induced platelet aggregation. PAR4 is cleaved by plasmin at the thrombin-cleavage site R47. Desensitization of PAR4 completely eliminates plasmin-induced aggregation. Platelets treated with a PAR4 antagonist do not aggregate in response to plasmin. Effects of Plasmin on von Willebrand Factor and Platelets van der Vorm et al. e224 plasmin in a canine animal model. 76 More in detail, plasmin activates protein kinase C (PKC) and stimulates dose-dependent diacylglycerol (and phosphatic acid) production by phospholipase C activation. 70 Subsequently, plasmin produces a small rise in platelet cytosolic Ca 2þ and a tyrosine kinase-dependent enhancement of Ca 2þ turnover. 70,71 Upon plasmin-induced platelet degranulation, ADP most likely plays a crucial role in the induction of platelet aggregation via the P2Tac receptor. 72 Interestingly, prolonged (>60 minutes) exposure of platelets to very low doses (0.05 CU/mL) of plasmin also leads to platelet aggregation and enhanced expression of procoagulant activity. 77 Likewise, upon lowering the temperature to 22°C, incubation with low concentrations of plasmin (<0.1 CU/mL) induces strong platelet aggregation. 78 These results may be of clinical relevance, because the fibrinolytic system was found to be activated during cardiopulmonary bypass, in which the temperature of the patient's blood is reduced. 78 Plasmin at concentrations less than 1.0 CU/mL can cause a dose-and time-dependent inhibition of platelet aggregation and thromboxane B2 (TXB 2 , inactive metabolite of TXA 2 ) production in response to thrombin, ionophore A23187, and collagen. 61,74,79,80 This plasmin-induced inhibitory effect may result from a proteolysis-mediated increase in cyclic AMP, 81 blockage of the mobilization of arachidonic acid from membrane phospholipid pools, 79 and/or modification of the expression of glycoprotein receptors (GPIb and GPIIb/IIIa, see below for more details). Interestingly, plasmin synergizes with prostaglandin I2 in inhibiting thrombin-and ADPinduced platelet activity, a finding of potential importance for the control of platelet accrual at sites of vascular injury, where the release of tPA and prostaglandin I2 by adjacent endothelial cells is generally increased. 82 Of note, in plasma, the main effect of plasmin is impairment of aggregation. 83,84 However, this is not related to a direct effect of plasmin on platelets. Plasmin degrades fibrinogen and fibrin/fibrinogen degradation products bind to fibrinogen and/or GPIIb/IIIIa receptors, both resulting in a reduction of the formation of fibrinogen bridges between platelets. 85 Additionally, in plasma, plasmin is inhibited by α2-antiplasmin. 86

Plasmin Activates Platelets via PAR4
Plasmin cleaves both thrombin receptors PAR1 and PAR4 on platelets. 87,88 Cleavage by plasmin was reported to be 1,700fold lower relative to thrombin cleavage. 89,90 Four cleavage sites have been identified on the PAR1 exodomain (R41, R70, K76, and K82). R41 is the thrombin-cleavage site and generates a transiently activated exodomain. The R70, K76, and K82 sites are located on a linker region that tethers the ligand to the body of the receptor. However, the high affinity of plasmin for R70, K76, and K82 and the low affinity for R41 (thrombin-cleavage site) result predominantly in inactivation of PAR1 by truncating the tethered ligand at the distal sites. 87 Additionally, desensitization of PAR1 had no effect on plasmin-induced platelet aggregation. 88 PAR4, on the other hand, is cleaved by plasmin at the thrombin-cleavage site R47. 88 As a result, desensitization of PAR4 completely elimi-nated aggregation in response to plasmin, and platelets treated with a PAR4 antagonist, as well as platelets isolated from PAR4 homozygous null mice, failed to aggregate in response to plasmin. Altogether, Quinton et al concluded that plasmin induces platelet aggregation primarily through slow cleavage of PAR4. 88

Plasmin Induces Reversible Translocation of Platelet GPIb
The addition of plasmin to washed platelets results in progressive loss of GPIb as measured by fluorescence flow cytometry and by the loss of ristocetin-induced VWF-dependent platelet agglutination. 91,92 The loss of platelet surface GPIb, initially assigned to plasmin-mediated cleavage of GPIb, 92-94 is now known to be related to a redistribution of GPIb, which moves from the external membrane into the surface-connected canalicular system. 95 This translocation of GPIb is related to actin polymerization, as cytochalasin D (a specific inhibitor of actin polymerization) inhibited the migration of GPIb after plasmin stimulation. 95 Subsequently, neutralization of plasmin by the inhibitors aprotinin and tripeptide Val-Phe-Lys-CH 2 Cl permitted a time-dependent recovery of platelet agglutination. 96 The functional recovery was accompanied by a restoration of a normal amount of GPIb on the platelet surface. This suggests a reverse translocation of the GPIb molecules initially present at the cell surface. 96

Effect of Plasmin on GPIIb/IIIa
Plasmin exerts opposing effects on GPIIb/IIIa, resulting in enhanced or impaired fibrinogen binding and platelet aggregation. On the one hand, plasmin-induced platelet activation and granule release result in the expression of active GPIIb/ IIIa receptors on the cell surface. 97 On the other hand, in plasma, plasmin degrades GPIIIa by cleaving a proteolytically susceptible loop domain, but only after degradation of the solubilized GPIIb/IIIa by Staphylococcus aureus V8 (Glu-C) endoprotease. 83 In contrast, treatment of isolated platelets with plasmin has no effect on GPIIb/IIIa expression. 95 These findings suggest that, in plasma, plasmin modifies GPIIIa by a unique proteolytic event that is dependent on fibrinogen binding and, consequently, is accompanied by significant reduction in fibrinogen binding and aggregation. 83

Conclusion
Although the mechanism of plasmin generation and its function in fibrinolysis are rather well-defined, the precise (patho) physiological effects of plasmin on other components of the coagulation cascade are hitherto not completely understood. In this review, we provide an overview of the available evidence on the effects of plasmin on VWF and platelets. Summarizing, it can be stated that, in addition to ADAMTS13, plasmin can regulate the thrombogenicity of VWF through its protease activity. This role of plasmin appears particularly important in pathologies in which high-molecular-weight VWF multimers accumulate, as exemplified by TTP. Moreover, although plasmin-mediated platelet activation is unlikely to play a significant role in normal hemostasis, during pharmacological thrombolysis where the plasmin activity could reach as high as 1 CU/mL, it may have implications in terms of vessel patency and effectiveness of treatment. Hence, future research should focus on obtaining a better understanding of the molecular mechanisms of plasmin binding to and cleavage of VWF as well as of plasmin's effects on platelet activation and aggregation. For experiments with platelets, particular attention should be given to simulating the in vivo environment as much as possible in the experimental conditions to obtain reliable results. Together, this knowledge on the interaction of plasmin, VWF, and platelets will support the ongoing development of novel therapies 98,99 as well as the use of existing thrombolytic therapy to potentially prevent the occurrence of, or degrade, existing VWF-platelet aggregates in conditions such as TTP.