Correction of haemorrhagic shock-associated coagulopathy and impaired haemostasis by plasma, prothrombin complex concentrates or an activated protein C-targeted DNA aptamer in mice.

Even with extensive transfusion support, trauma-induced bleeding often leads to death. Early intervention may improve outcomes, yet which blood products, factor concentrates, or other drugs constitute optimal treatment is unclear. Patients with acute traumatic coagulopathy (ATC), arising from trauma and haemorrhagic shock, have the worst prognosis. Here, multiple interventions were compared in a mouse model of ATC. After the trauma of tissue excision, anaesthetized mice were bled to 35 mm Hg mean arterial pressure, maintained under shock for 60 min, and resuscitated with fluids equal in volume to the shed blood. Resuscitated mice were subjected to liver laceration to test haemostasis and blood loss was quantified. Saline-treated mice lost two- to three-fold more blood than sham-treated animals and were coagulopathic by prothrombin time elevation post- versus pre-procedure. Murine fresh-frozen plasma (mFFP), anti-activated protein C aptamer HS02-52G, or prothrombin complex concentrates eliminated the bleeding diathesis and coagulopathy; fibrinogen, plasminogen activator inhibitor-1, or tranexamic acid ameliorated bleeding or coagulopathy, but not both. HS02-52G and mFFP also eliminated the changes in plasma aPC and tissue plasminogen activator levels observed in saline-treated mice, as judged via microtiter plate biomarker assays. Procoagulant interventions, especially inhibiting aPC, could be beneficial in human ATC.

CD248 enhances tissue factor procoagulant function, promoting arterial and venous thrombosis in mouse models.

BACKGROUND: CD248 is a pro-inflammatory, transmembrane glycoprotein expressed by vascular smooth muscle cells (VSMC), monocytes/macrophages, and other cells of mesenchymal origin. Its distribution and properties are reminiscent of those of the initiator of coagulation, tissue factor (TF). OBJECTIVE: We examined whether CD248 also participates in thrombosis. METHODS: We evaluated the role of CD248 in coagulation using mouse models of vascular injury, and by assessing its functional interaction with the TF-factor VIIa (FVIIa)-factor X (FX) complex. RESULTS: The time to ferric chloride-induced occlusion of the carotid artery in CD248 knockout (KO) mice was significantly longer than in wild-type (WT) mice. In an inferior vena cava (IVC) stenosis model of thrombosis, lack of CD248 conferred relative resistance to thrombus formation compared to WT mice. Levels of circulating cells and coagulation factors, prothrombin time, activated partial thromboplastin time, and tail bleeding times were similar in both groups. Proximity ligation assays revealed that TF and CD248 are <40 nm apart, suggesting a potential functional relationship. Expression of CD248 by murine and human VSMCs, and by a monocytic cell line, significantly augmented TF-FVIIa-mediated activation of FX, which was not due to differential expression or encryption of TF, altered exposure of phosphatidylserine or differences in tissue factor pathway inhibitor expression. Rather, conformation-specific antibodies showed that CD248 induces allosteric changes in the TF-FVIIa-FX complex that facilitates FX activation by TF-FVIIa. CONCLUSION: CD248 is a newly uncovered protein partner and potential therapeutic target in the TF-FVIIa-FX macromolecular complex that modulates coagulation.

Sustained depletion of FXIII-A by inducing acquired FXIII-B deficiency.

The activated form of coagulation factor XIII (FXIII-A2B2), FXIII-A*, is a hemostatic enzyme essential for inhibiting fibrinolysis by irreversibly crosslinking fibrin and antifibrinolytic proteins. Despite its importance, there are no modulatory therapeutics. Guided by the observation that humans deficient in FXIII-B have reduced FXIII-A without severe bleeding, we hypothesized that a suitable small interfering RNA (siRNA) targeting hepatic FXIII-B could safely decrease FXIII-A. Here we show that knockdown of FXIII-B with siRNA in mice and rabbits using lipid nanoparticles resulted in a sustained and controlled decrease in FXIII-A. The concentration of FXIII-A in plasma was reduced by 90% for weeks after a single injection and for more than 5 months with repeated injections, whereas the concentration of FXIII-A in platelets was unchanged. Ex vivo, crosslinking of α2-antiplasmin and fibrin was impaired and fibrinolysis was enhanced. In vivo, reperfusion of carotid artery thrombotic occlusion was also enhanced. Re-bleeding events were increased after challenge, but blood loss was not significantly increased. This approach, which mimics congenital FXIII-B deficiency, provides a potential pharmacologic and experimental tool to modulate FXIII-A2B2 activity.

Proteolytic modulation of factor Xa-antithrombin complex enhances fibrinolysis in plasma.

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~13kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys-Met339 and Lys-Asp389. Ligand blots demonstrated (125)I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.

Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway.

The coagulation and complement pathways simultaneously promote homeostasis in response to injury but cause tissue damage when unregulated. Mechanisms by which they cooperate are poorly understood. To delineate their interactions, we studied the effects of thrombin and C5 convertase on C5 in purified and plasma-based systems, measuring release of the anaphylatoxin C5a, and generation of C5b, the initial component of the lytic membrane attack complex. Thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b. However, thrombin efficiently cleaved C5 at a newly identified, highly conserved R947 site, generating previously undescribed intermediates C5(T) and C5b(T). Tissue factor-induced clotting of plasma led to proteolysis of C5 at a thrombin-sensitive site corresponding to R947 and not R751. Combined treatment of C5 with thrombin and C5 convertase yielded C5a and C5b(T), the latter forming a C5b(T)-9 membrane attack complex with significantly more lytic activity than with C5b-9. Our findings provide a new paradigm for complement activation, in which thrombin and C5 convertase are invariant partners, enhancing the terminal pathway via the generation of newly uncovered C5 intermediates. Delineating the molecular links between coagulation and complement will provide new therapeutic targets for diseases associated with excess fibrin deposition and complement activation.

Enhanced fibrinolysis by proteolysed coagulation factor Xa.

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaalpha) must be sequentially cleaved by plasmin or autoproteolysis, producing FXabeta and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaalpha/beta or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaalpha/beta to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaalpha active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaalpha/beta or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaalpha/beta or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaalpha undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.

Alpha1-antitrypsin suppresses TNF-alpha and MMP-12 production by cigarette smoke-stimulated macrophages.

We have previously observed that mice exposed to cigarette smoke and treated with exogenous alpha(1)-antitrypsin (A1AT) were protected against the development of emphysema and against smoke-induced increases in serum TNF-alpha. To investigate possible mechanisms behind this latter observation, we cultured alveolar macrophages lavaged from C57 mice. Smoke-conditioned medium caused alveolar macrophages to increase secretion of macrophage metalloelastase (MMP-12) and TNF-alpha, and this effect was suppressed in a dose-response fashion by addition of A1AT. Macrophages from animals exposed to smoke in vivo and then lavaged also failed to increase MMP-12 and TNF-alpha secretion when the animals were pretreated with A1AT. Because proteinase activated receptor-1 (PAR-1) is known to control MMP-12 release, macrophages were treated with the G protein-coupled receptor inhibitor, pertussis toxin; this suppressed both TNF-alpha and MMP-12 release, while a PAR-1 agonist (TRAP) increased TNF-alpha and MMP-12 release. Smoke-conditioned medium caused increased release of the prothrombin activator, tissue factor, from macrophages. Hirudin, a thrombin inhibitor, and aprotinin, an inhibitor of plasmin, reduced smoke-mediated TNF-alpha and MMP-12 release, and A1AT inhibited both plasmin and thrombin activity in a cell-free functional assay. These findings extend our previous suggestion that TNF-alpha production by alveolar macrophages is related to MMP-12 secretion. They also suggest that A1AT can inhibit thrombin and plasmin in blood constituents that leak into the lung after smoke exposure, thereby preventing PAR-1 activation and MMP-12/TNF-alpha release, and decreasing smoke-mediated inflammatory cell influx.

Plasminogen binds to plasmin-modulated factor Xa by Ca(2+) - and C-terminal lysine-dependent and -independent interactions.

Plasminogen binding to receptors involves both C-terminal lysine- dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca (2+) -enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca (2+) may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1 - 77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 2:1 plasminogen:FXa33/13 stoichiometry for both, which were proportionately enhanced by Ca (2+) . Only Lys-Pg demonstrated significant Ca (2+) -independent binding to FXa33/13. In the presence of Ca (2+) , weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1 - 3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1 - 3 binding to FXa33/13 had both Ca (2+) -dependent and -independent components. The principal K (d) for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca (2+) , eACA inhibited FXa33/13 binding to K1 - 3 by 30%, but eliminated binding in the absence of Ca (2+) . These studies suggest that Ca (2+) -dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1 - 77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.