Systemic Vascular Basement Membrane Markers Linked to Synovial Vascular Remodeling Are Bio-Markers of Hemarthrosis in Patients with Hemophilia

Introduction: Acute painful joint episodes in hemophilia are not always associated with hemarthrosis. Point-of-Care Musculoskeletal Ultrasound with Power Doppler (POC MSKUS/PD) is a fast and sensitive imaging modality to determine the presence of hemarthrosis and direct management. Systemic collagen cleavage products reflecting extracellular matrix, bone and soft tissue turnover may serve as an alternative tool for the detection of hemarthrosis. Based on previous observations of pronounced vascular remodeling within the synovial tissue during hemarthrosis in patients and hemophilic mouse models, we hypothesized that cleavage products of collagen IV, found in the basement membrane of blood vessels may differentiate between bleeding and non-bleeding states.

Methods: Joint bleeding was induced in FVIII^-/- mice (+/- FVIII-treatment before and shortly after injury). Synovial vascular remodeling was assessed 14 days later by MSKUS/PD and histology including αSmooth Muscle Actin (αSMA)-staining. Murine plasma samples were analyzed for C4M2 and P4NP-7S, which are type IV collagen turnover products. To determine human relevance, 31 adult patients with hemophilia (PWH), ≥ age 21, were studied prospectively for 3 years with MSKUS/PD during pain-free intervals and painful events for bleed status, vascular flow and 11 plasma markers of collagen turnover(C2M, ARGS, C3M, Pro-C3, C5M, Pro C5, C4M2, P4NP-7S, Col18N, DCN- CS, ColNeo10, NIC).

Results: Type IV collagen turn-over markers were significantly elevated in plasma of FVIII^-/- deficient mice 14 days after joint bleeding when compared to baseline (C4M2: 2.575 ng/mL vs. 1.94 ng/mL, p=0.029 and P4NP-7S: 80.3 ng/mL vs. 67.81 ng/mL, p= 0.0152 respectively ). This increase coincided with a significant increase in vessel number (2.3 fold, p=0.0008), vascular flow by PD (2.3 fold, p= 0.014) and αSMA staining (5.1 fold, p= 0.0007). C4M2 and P4NP7S plasma levels correlated negatively with αSMA staining, suggesting a role for αSMA in vessel stabilization (C4M2 p=0.02, Pearson r= -0.5707; P4NP-7S p= 0.0130, Pearson r= -0.6238). FVIII-treatment reduced plasma C4M2 and P4NP-7S levels to baseline (C4M2 1.98 ng/mL vs. 1.94 ng/mL, P4NP-7S 59.98 ng/mL vs. 67.81 ng/mL), but had no effect on the other vascular parameters. In patients, results from 91 visits were compiled. Twenty five were due to acute painful episodes with 16 confirmed hemarthroses. Of the 11 biomarkers evaluated, only C4M2 and P4NP-7S were transiently but significantly elevated in plasma during joint bleeding compared to pain free intervals or to non-bleeding painful episodes. Plasma C4M2 levels during acute painful bleeding episodes when compared to non-bleeding states were 41.65 vs. 29.69 ng/mL (p=0.0003), and P4NP-7S levels were 418.9 vs 278.1 ng/mL (p=0.0007), respectively. Baseline levels of C4M2 and P4NP-7S in PWH were above the lab reported normal range, indicating ongoing high vascular basement membrane turnover. Additionally, PWH who developed joint bleeding episodes had a higher level of collagen IV biomarkers at baseline when compared to PWH who did not experience joint bleeding (C4M2 33.91 vs 29.07 ng/mL, P4NP7s 320.6 vs 292.8 ng/mL), although this was not statistically significant. Hemarthrosis was accompanied by pronounced abnormal synovial microvascular flow evidenced by PD, while plasma levels of both biomarkers correlated positively with joint PD signal (C4M2, p= 0.0400, Pearson r=0.4409; P4NP7S-p=0.0141, Pearson r=0.5154).

Conclusions: Our findings suggest that systemic vascular collagen turn-over products may be novel biomarker candidates to identify patients at high risk for joint bleeding and to diagnose acute hemarthrosis. Hemarthrosis in hemophilia is associated with pronounced synovial vascular remodeling. Cleavage products of type IV collagen, but not of any other collagen, were systemically elevated in PWH and distinguished bleeding from non-bleeding painful episodes. Type IV collagen is found exclusively in basement membranes and in the case of joint bleeding, the presumed source of these turnover markers is newly formed synovial blood vessels. Therefore, our findings support the concept that vascular instability during neovascularization is involved in the dynamics of hemophilic joint bleeding. These findings broaden the scope of potential diagnostic tools substantially, opening new avenues for personalized treatment of PWH.