Fig 2: Injection of Sema7a results in increased MIRI and PNC formation.Mice were injected with either recombinant semaphorin 7a (rmSema7a) or Fc control (rmIgG2A Fc) and then subjected to 1 h of ischemia followed by 2 h reperfusion. Samples were taken after 1 or 120 min reperfusion. a Representative TTC-stained slices of myocardial tissue showing infarcted area (blue/dark = retrograde Evans blue staining; red and white = AAR, white = infarcted tissue; 120 min) with b systematic evaluation of infarct sizes and corresponding troponin I plasma levels (120 min, n = 6/group). c Representative histological sections (scale bar 100 µm) of WT animals injected with either IgG Fc control or rmSema7a (120 min) and d number of PNCs counted in myocardial tissue sections of AAR in animals (120 min, n = 9/group). e Representative flow-cytometric plots of PNCs in the blood of sham animals, rmSema7a- or IgG Fc control–treated mice, showing GPIb (CD42b) and P-selectin (CD62P) after 1 and 120 min reperfusion. Systematic evaluation of flow-cytometric expression of mean fluorescence intensity (MFI) for f GPIb (CD42b, n = 6;6;4;4;6) and g P-selectin (CD62P, n = 6;3;5;3;7) expression. h Systematic evaluation of PNCs in % by flow cytometry in the blood of animals injected with rmSema7a or Fc control at 1 and 120 min (n = 6;4;4;4;5). i Representative flow-cytometric plots of PNCs in the AAR showing the presence of GPIb (CD42b) and P-selectin (CD62P) after 1 and 120 min reperfusion. Systematic evaluation of flow-cytometric expression of MFI for j GPIb (CD42b, n = 5;5;5;4;6) and k P-selectin (CD62P, n = 6;4;5;3;5) and l systematic evaluation of PNCs in % by flow cytometry in the AAR of animals injected with rmSema7a or Fc control at 1 and 120 min (n = 5;4;5;5;5). Comparisons in b and d were analyzed by unpaired two-tailed Student’s t-tests (data are mean ± SD). For f–h, j–l we used log transformation of data to conform normality. For log-transformed data, unpaired two-tailed Student’s t-tests were performed on the log values and results are displayed as geometric means and their 95% confidence intervals. *p < 0.05, **p < 0.01, and ***p < 0.001 as indicated.

Fig 3: Anti-Sema7a reduces MIRI, platelet activation and PNC formation.Animals were injected with either anti-semaphorin 7A antibody (anti-Sema7a) or IgG control 5 min before starting 120 min reperfusion after 60 min of ischemia, with samples taken after 1 or 120 min of reperfusion. a Representative TTC-stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR, white = infarcted tissue) with b systematic evaluation of infarct sizes (n = 6/group) and correlating troponin I plasma levels (n = 6;8). c Representative histology sections (scale bar 100 µm) of WT animals injected with either IgG control or anti-Sema7a and d number of PNCs counted from myocardial tissue sections (n = 9/group). e Representative flow cytometry plots of PNCs in the blood of Sham, anti-Sema7a-injected or IgG control-injected animals, expressing GPIb (CD42b) and P-selectin (CD62P). Systematic evaluation of flow-cytometric expression of mean fluorescence intensity (MFI) for f GPIb (CD42b, n = 5;4;3;4;5) and g P-selectin (CD62P, n = 6;4;4;3;5) and h systematic evaluation of PNCs in % by flow cytometry in the blood of animals injected with anti-Sema7a or IgG control at 1 and 120 min (n = 3;4;4;3;4). i Systematic evaluation of flow-cytometric expression of MFI for j GPIb (CD42b n = 4;5;3;3;4) k P-selectin (CD62P, n = 4/group) and l systematic evaluation of PNCs in % by flow cytometry in the AAR of animals injected with anti-Sema7a or IgG control at 1 and 120 min (n = 5;6;5;4;4). Comparisons in b, d were analyzed by unpaired two-tailed Student’s t-tests (data are mean ± SD). For f–l we used log transformation of data to conform normality. For log-transformed data, unpaired two-tailed Student’s t-tests were performed on the log values and results are displayed as geometric means and their 95% confidence intervals. *p < 0.05, **p < 0.01, and ***p < 0.001 as indicated.

Fig 4: Patients with acute myocardial infarction show increased PNCs and plasma SEMA7A.Samples were taken from patients with acute myocardial infarction, patients undergoing cardiac surgical operations with (HLM) or without cardiopulmonary bypass (Off-Pump) and healthy controls. a Flow-cytometric evaluation of PNCs’ expression of GPIb (CD42b) in the presented patient groups (Healthy, MI, and HLM n = 7; Off-pump n = 8). b Flow-cytometric evaluation of PNCs for CD62P expression on neutrophils (n = 4;5;5;5). c PAC-1 binding to PNCs (n = 4;8;7;6). d Representative flow-cytometric histograms of groups measured and e Sema7a in the plasma of patients with acute MI (n = 10), patients undergoing cardiac surgical operations with (HLM) or without cardiopulmonary bypass (Off-Pump) and healthy controls (all n = 4/group). For comparison we performed one-way analyzes of variance followed by Dunnett’s tests to group myocardial infarction. Data are mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001 as indicated.

Fig 5: Schematic drawing of the role of Sema7a during myocardial IR.a During myocardial ischemia shear stress and hypoxia result in cleavage of Sema7a from the surface of erythrocytes as the main source of Sema7a within the vascular bed. b The released Sema7a then engages the Glycoprotein Ib receptor and activates platelets which then exposes the integrin receptors resulting in platelet–neutrophil complex formation (PNCs). c Activated platelets and PNCs migrate from the vasculature to the ischemic tissue which results in tissue injury and destruction.