Normally factor (F) VIII is not expressed in megakaryocytes, but when human FVIII was transgenically expressed in murine megakaryocytes, it was stored in platelet ␣-granules and released at sites of injury. This platelet FVIII (pFVIII) is effective in correcting hemostasis, even in the presence of circulating inhibitors, so it offers a potential gene therapy strategy for hemophilia A. To understand clot development by pFVIII, we have examined clot response to laser injury in both cremaster arterioles and venules in FVIII null mice either infused with FVIII or transgenic for pFVIII. In both sets of vessels, pFVIII is at least as effective as infused FVIII. However, there are temporal and spatial differences in fibrin and platelet accumulation within clots depending on how FVIII is delivered. These differences may be related to the temporal and spatial distribution of the ␣-granular-released FVIII within the developing clot, and may explain the increased frequency and size of embolic events seen with pFVIII. These observations may not only have implications for the use of pFVIII in gene therapy for hemophilia A, but may also have physiologic consequences, explaining why many procoagulant factors are delivered both in the plasma and in platelet ␣-granules. (Blood. 2008;112:1101-1108) IntroductionWe and others have shown that human B-domainless factor (F) VIII can be expressed in murine megakaryocytes, stored in platelet ␣-granules independent of von Willebrand factor (VWF), and released at sites of injury in hemophilia A FVIII null mice. [1][2][3] Correction of bleeding occurred in FVIII null mice transgenic for platelet-specific FVIII (pFVIII) expression, in spite of there being no detectable plasma FVIII level. In addition, pFVIII is protected from circulating inhibitors, 3,4 a significant problem in the hemophilia A population. 5 Several editorials have, therefore, suggested that this pFVIII gene therapy strategy may be of benefit in hemophilia A patients with problematic inhibitors who do not respond to present-day therapies to eliminate such inhibitors. 6,7 Aside from a megakaryocyte-specific gene therapy, all other FVIII-based gene therapy strategies for hemophilia A correct plasma FVIII levels. 8 We believe that the details of how FVIII released from activated platelets affects clot development must be examined to be assured that there are no untoward side effects before clinical application. Several clotting models have been examined in FVIII null mice, which suggest that pFVIII is at least as efficacious as plasma FVIII. The tail exsanguination model, which involves both arterials and venules of various sizes and which extends over 16 hours, suggested very high relative efficacy for pFVIII, but we have shown that this model may be too sensitive to pFVIII, likely due to hypovolemia with subsequent blood stasis in the tail. 1 A cuticular bleed model, which also involves both arterial and venule injury and which extends over 8 hours, suggested less relative efficacy. 1 Since both the tail exsanguination and c...

Ectopically expressed, human B-domainless (hB) factor 8 (F8) in platelets improves hemostasis in hemophilia A mice in several injury models. However, in both a cuticular bleeding model and a cremaster laser arteriole/venule injury model, there were limitations to platelet-derived (p) hBF8 efficacy, including increased clot embolization. We now address whether variants of F8 with enhanced activity, inactivation resistant F8 (IR8) and canine (c) BF8, would improve clotting efficacy. In both transgenic and lentiviral murine model approaches, pIR8 expressed at comparable levels to phBF8, but pcBF8 expressed at only approximately 30%. Both variants were more effective than hBF8 in cuticular bleeding and FeCl 3 carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants. (Blood. 2010;116(26): 6114-6122) IntroductionHemophilia A is an X-chromosome-linked bleeding disorder due to a deficiency in clotting factor VIII (F8), affecting approximately 1:5000 males. 1,2 In this country, significant hemophilia A bleeding episodes are primarily treated by infusions of recombinant F8; however, limitations result from F8's short half-life, 3 the high cost of the replacement factor, 4 and clinically relevant inhibitor development to F8 in 20%-30% of patients. 5 Several studies have focused on modulating F8 hemostasis using therapeutic strategies such as the attachment of recombinant F8 to pegylated liposomes. 6 Bypass products of either activated prothrombin complex concentrates or activated recombinant F8 have been successfully used in patients with inhibitors. [7][8][9] These alternate approaches to F8 therapy do not provide continuous coverage, and may not always be effective. 8,9 Gene therapy for F8 replacement is attractive as there is a wide therapeutic window for F8 corrective plasma levels. 10 Past gene transfer studies have focused on liver expression of hF8; however, sustained high F8 expression levels have proven difficult to achieve in these studies. [11][12][13] One approach to improve outcome in these studies has been to increase the efficacy of F8 by designing variants of F8 with improved in vitro activity. 14 Inactivation resistant F8 (IR8) is one such variant and has increased resistance to thrombin and activated protein C inactivation in vitro. 15 However, this variant also has a decreased von Willebrand factor (VWF) binding, which may limit its plasma half-life and clinical utility. 16 Previous work has demonstrated that targeted deli...

To cite this article: Gewirtz J, Thornton MA, Rauova L, Poncz M. Platelet-delivered factor VIII provides limited resistance to anti-factor VIII inhibitors. J Thromb Haemost 2008; 6: 1160-6. Summary. Background: Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti-FVIII inhibitors. Objective: We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIII null mouse in the presence of circulating inhibitors. Methods: FVIII null mice that were transgenic for pFVIII (pFVIII/FVIII null ) were compared with FVIII null mice receiving infused FVIII in a FeCl 3 carotid injury model in the presence of anti-FVIII inhibitors. Results: After injury, pFVIII/FVIII null mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia. Conclusion: In FVIII null mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of 6-fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail-clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail-vein exsanguination model to low levels of pFVIII.

Gene therapy strategies directed at expressing Factor (F) VIII in megakaryocytes may have several potential advantages in the treatment of severe hemophilia A. Among these is that platelet (p) FVIII may be protected from circulating inhibitors. We have previously described a murine transgenic line that expressed human B-domainless FVIII in a megakaryocyte-specific fashion and that this pFVIII was localized to within alpha granules. We also showed that these platelets contained FVIII equivalent to an infusion of 9% human FVIII into FVIIInull mice. We further showed that these pFVIII mice, on a FVIIInull murine background, formed stable clots in a FeCl3 carotid artery injury model. We then tested the ability of infused anti-human FVIII inhibitors in this setting. Using up to 100 μL of ESH8 monoclonal antibody (Ab) to the FVIII C2 light chain (1 μg/mL), anti-human polyclonal Ab (11 mg/mL) or a monoclonal Ab to the A2 domain (5 mg/mL), we were unable to alter thrombus formation in the carotid artery model. However, by using a 1:1:1 mixture of these inhibitors, we were able to show a dose-response curve. None of these mice developed thrombocytopenia suggesting that pFVIII is not exposed on the surface of circulating platelets. We then compared these studies to an acute infusion of the same inhibitor mixture in a FVIIInull mice receiving a 25% hFVIII correction. These studies showed that pFVIII/FVIIInull mice were ∼100-fold more resistant to inhibitors than plasma FVIII infusion into a FVIIInull mice in the carotid artery injury model. Since we had shown in the pFVIII mice that the FVIII is stored in alpha-granules, which can also store circulating Ab, we wondered whether the pFVIII/FVIIInull mice would be more sensitive to inhibitors when exposed in a chronic model where animals receive repeat doses of the inhibitor mixture. We therefore infused 3 doses of the inhibitor over 10 days, measured plasma and platelet inhibitor levels, and found that despite detectable stores of inhibitor within their platelets, these mice still demonstrated a comparable ability to form thrombosis as mice in the acute model with comparable plasma inhibitor levels. These studies suggest that pFVIII provides limited improved protection in mice with inhibitors comparable to <6 Bethesda Units (BU) in both acute and chronic models of inhibitor. Also, the presence of inhibitors within alpha-granules does not significantly inhibit the ability of these mice to form a clot. Our findings differ from a recent report of the efficacy of pFVIII in a tail vein survival model where pFVIII effectively protected mice from exsanguinations even in the presence of >100 BU/mL. We propose that the difference in outcome is due to the tail model being extremely sensitive to even low levels of pFVIII as exsanguinated, hypovolemic mice likely shunt blood away from their tail veins, and platelet activation and granular release are occurring in a low flow setting, while the FeCl3 model used in this report requires a plasma equivalency of >3–5% human FVIII to show even partial correction.

Vascular damage due to trauma or disease exposes circulating platelets to collagen in the subendothelial matrix. This is a critical event in the formation of a hemostatic plug or an occluding thrombus because collagen is not only a substrate for platelet adhesion but is also a strong platelet agonist. Platelets possess two physiologic collagen receptors: glycoprotein VI, a member of the immunoglobin superfamily, and the integrin α2β1. To design small molecule inhibitors of the interaction of platelets with collagen, we focused on α2β1 as a target because murine models of α2β1 deficiency display normal bleeding times and only a slight decrease in platelet activation by collagen and because the small number of reported patients with congenital α2β1 deficiency demonstrated only a mild bleeding diathesis. Thus, α2β1 antagonists could be effective anti-thrombotic agents with minimal toxicity, especially when combined with other anti-platelet drugs. We have developed a class of compounds that target the I-like domain of the β1 subunit, an allosteric site that regulates collagen binding to α2β1 by preventing the conversion of α2β1 from an inactive (low affinity) to an active (high affinity) conformation. This class of compounds is based on a proline-substituted 2,3-diaminopropionic acid scaffold. Structure-activity relationship studies of the scaffold have focused on optimization of the proline moiety, the urea functionality, and the sulfonyl group and have resulted in the development of potent inhibitors of α2β1-mediated platelet adhesion to collagen with IC50’s in the high picomolar to low nanomolar range. In particular, optimization of the proline moiety lead to compounds with high potency: transitioning from proline (DB496, IC50 of 29–62 nM) to a thiazolidine (SB68A) improved the IC50 to 2–8 nM; adding a methyl group at the 2 position of the thiazolidine (SB68B) slightly improved the IC50 to 1–12 nM; adding two methyl groups at the 5 position of the thiazolidine (SW4-161) resulted in a lead compound with an IC50 of 0.33–8 nM. As expected, the compounds had no effect on the binding of isolated α2 I-domains to collagen, consistent with their I-like domain mode of activity. Further, they were specific for α2β1-mediated platelet adhesion to collagen because they had no impact on ADP-stimulated platelet aggregation when added at 2 μM, a concentration more than 100-fold greater than the IC50 for inhibition of platelet adhesion to collagen. The compounds were also strong inhibitors of murine platelet adhesion to collagen and when tested in the ferric chloride-initiated murine carotid artery injury model, displayed activity similar to aspirin. Thus, 71% of untreated mice in this thrombosis model developed occlusive thrombi that remained stable for the 30 min duration of the assay, whereas stable thrombi developed in only 32% of mice treated with 1g/kg aspirin orally and in 41% of mice receiving 60 mg/kg CSW4-161intravenously. In summary, we have developed a class of potent inhibitors of the integrin α2β1 that demonstrate both in vitro and in vivo anti-platelet activity. Further development of this class of compounds may result in novel and relatively non-toxic anti-thrombotic agents.