**3.1. Hemophilia a and factor VIII**

Hemophilia A is majorly caused by deficiency in FVIII antigen levels or mutations in FVIII gene that effect FVIII functions [18, 19]. FVIII is encoded by the gene that localized on the long arm of X chromosome and the gene consists of 26 exons [18, 19]. A total of 2537 mutations are identified on FVIII gene [20]. FVIII is highly expressed in the liver [21, 22]. The mature FVIII protein consists of 2332 amino acids with 6 domains. These domains include A1, A2, B, A3, C1 and C2 [23, 24]. In the blood FVIII is activated by thrombin or FXa [25, 26]. Thrombin cleaves FVIII at R372, R740 and R1689 and removes B domain [26]. Similarly, FXa cleaves FVIII at K36, R336, R562, R740, R1689 and R1721 [27]. FXa mediated cleavage of FVIII at K36 and R336 leads to inactivation of FVIIIa [27]. APC inactivates FVIII by proteolytically cleaving FVIIIa at R336 and R562 which leads to destabilization of A1 and A2 domain interaction [28, 29] (**Figure 2**). In 1960s, major treatment for hemophilia A is whole blood or plasma transfusion [30, 31]. This treatment has a drawback of viral transfusion along with the coagulation factors. Treatments of mild hemophilia A include vasopressin analogs to enhance the synthesis of FVIII, 1-Desamino-8d-arginine vasopressin (DDAVP) is a vasopressin analog clinically used to enhance the plasma levels of FVIII [32, 33]. Recombinant FVIIa and FVIII are also used to prevent bleeding events in the hemophilia patients [34].These clotting factors are also supplemented with FVIIa or factor FVII inhibitor bypassing agent (FEIBA) to enhance the function of FVIIa and FVIII, whereas FEIBA enhanced the risk of thrombosis [35, 36]. Recent studies elucidated that stabilized recombinant FVIII can be used as a therapeutic for hemophilia A, this includes more stable isoforms of FVIII such as B domain deleted FVIII (BDD FVIII) [37] (**Table 1**). The ongoing research is focusing on using BDD FVIII as a gene therapy by incorporating it into the viral vectors and delivering it into the patient [38].

mild (5–40%) [48]. Current therapies for hemophilia B include plasma derived FIX, recombinant FIX, recombinant FIX fused with polyethylene glycol (PEG), recombinant FIX fused with Fc portion of immunoglobulin G, FIX fused with albumin mutant FIX [48]. The disadvantages of plasma derived FIX is that it has a very short half-life in the patient plasma and plasma derived FIX has chances of viral contamination. Recombinant FIX is produced in Chinese hamster ovary cells [48]. rFIX has an increased half-life compared to plasma derived FIX, it over came the problem of viral contamination, however rFIX showed 30% less activity compared to plasma derived FIX, due to variations in the glycosylation. Conjugation of FIX with PEG is known as PEGylation. PEG serves as a shield for PEGylated FIX and protects it from proteolytic cleavage. PEGylated FIX's half-life is five times in mice compared to the half-life of rFIX [48]. FC fused FIX has a half-life of 48 h. The other FIX fusion protein in clinical trial is FIX fused with albumin. Single amino acid mutation in the catalytic domain of FIX (R338L) increased its Tenase activity by 2 fold and thrombin generation activity by 6 fold, therefore by

**FVIII-product Half-life in hours**

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Full length Plasma derived [31] 14.8–17.5 Plasma derived FVIII-vWF complex [39] 12.2–17.9 Recombinant Full length FVII [34] 14.6 ± 4.9 B-Domain Deleted FVIII [34] 14.5 ± 5.3 BDD-PEGylated [40] 14.69 ± 3.79 BDD-rFVIII-Fc [41] 19.7 ± 2.3 BDD-rFVIII EHL single chain [30] 14.2

Hemophilia C is caused by FXI deficiency where factor XI activity of 15–20 U/dL or lower.

Platelets are key components of primary coagulation system [50]. TF released from the damaged endothelial cells activates the platelets [51].Activated platelets get adhere to the site of damage with help of vWF [52]. Upon activation platelets expose phosphatidylserine which gives the lipid surface to the coagulation cascade [51]. Recent studies elucidate that platelets can play a major role in hemophilia, as hemophilia patients with same FIX or FVIII antigen levels has different clotting time due to variations in the platelet mediated coagulation activity [53–55]. Platelets store FVIII in the alpha granules therefore, platelets are being used as therapeutic components for hemophilia treatment, majorly in gene therapy. In a recent study, hemophilic dogs were transfused with genetically modified platelets (which can over express FVIII gene). Bleeding events were stopped

Surprisingly FXI deficiency does not show a severe bleeding phenotype [16].

in the hemophilic dogs after transfusing them with genetically altered platelets [53].

FIX R338L usage in gene therapy is under investigation [49].

**Table 1.** Development of FVIII therapy for hemophilia.

**3.3. Hemophilia C**

**4. Platelets in hemophilia**
